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.     

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.     

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).     

Interaction of copper-metallothionein from the American lobster, Homarus americanus, with glutathione

Organisms have harnessed the unique chemistry of copper for a variety of purposes. However, that same chemistry makes this essential metal toxic at elevated concentrations. Metallothioneins (MTs), a family of small metal-binding proteins, are thought to play a crucial role in the regulation of this reactive ion. Here we report that copper-metallothioneins from the American lobster, Homarus americanus, interact with the tripeptide glutathione (gamma-Glu-Cys-Gly). Glutathione in the cytosolic fraction prepared from the digestive gland of the American lobster coelutes with copper-metallothionein during size-exclusion chromatography. The latter protein can be separated into three isoforms by anion-exchange chromatography. All three isoforms belong to the class I MTs. CuMT-I and -II are very similar, whereas CuMT-III is distinct from isoforms I and II. The interaction between glutathione and MT isoforms was examined by ultrafiltration experiments and size-exclusion HPLC. CuMT-III forms a stable 1:1 complex with glutathione, with a dissociation constant of 1 microM. CuMT-I/II makes a transient complex with glutathione, which releases copper as a copper-glutathione complex. This complex can function as the source of Cu(I) in the restoration of the oxygen-binding capacity of copper-free hemocyanin. These studies suggest that metallothionein and glutathione are intricately linked in the biochemistry of copper regulation.     

A mycosis of the American lobster, Homarus americanus, caused by Fusarium sp.

Successful inoculations of citrus red mites, Panonychus citri, with the nonoccluded virus were made by feeding healthy mites through membranes on contaminated lemon fruit. Manipulation of the membranes showed that healthy mites pick up the virus from contaminated surface substrates rather than from within plant cells. Electron microscopical studies substantiated that feces of infected mites should be highly infectious since the hindgut contains numerous virus rods.     

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.     

Phagocytosis as a cellular immune response mechanism in the American lobster, Homarus americanus.

Procedures to quantitate accurately the in vitro phagocytosis of sheep erythrocytes and Aerococcus viridans var. homari (formerly Gaffkya homari) bacteria by hemocytes of the American lobster were utilized to assess the effect of various vaccines on the humoral and cellular defense mechanisms of the lobster. Both the percent of hemocytes showing phagocytosis and the number of particles phagocytosed increased markedly in many of the animals injected with Pseudomonas perolens cells or endotoxin. A lesser response was elicited by A. viridans cellular vaccines. In vivo phagocytosis was observed in the circulatory system of lobsters infected with A. viridans var. homari.Natural hemolymph agglutinins for sheep or rabbit erythrocytes were not affectd by any vaccines. Hemocyte numbers dropped initially after administration of P. perolens endotoxin or cells. The bactericidin level was enhanced in samples taken from vaccinated animals.     

Structural and functional diversity of copper-metallothioneins from the American lobster Homarus americanus

The role of copper metallothionein (CuMT) in copper metabolism and metalloenzyme activation is poorly understood. We have chosen marine crustaceans, in which a direct correlation exists between levels of Cu(I)MT and Cu(I)-hemocyanin during the molt cycle (Engel and Brouwer, Biol. Bull. 173, 239-251, 1987) as unique model systems to study the involvement of MTs in metalloprotein activation and degradation. We have isolated three low-molecular weight, cysteine-rich copper proteins from the American lobster Homarus americanus, which we designate as CuMT-1, CuMT-2, and CuMT-3, respectively. As a first attempt to fully characterize these proteins, we have determined the sequence of the first 56 amino acids of CuMT-1. The results show this protein to belong to the class I MTs, i.e., related in primary structure to equine renal MT. CuMT-1 cannot transfer its copper to copper-depleted apohemocyanin. CuMT-2 belongs to the same class of MTs as CuMT-1, but CuMT-3 does not. The latter can reactivate lobster hemocyanin containing reduced amounts of Cu(I). Spectroscopic studies show that Cu(I) transfer from CuMT-3 to apohemocyanin initially results in the formation of distorted binuclear-copper sites, which subsequently slowly return to their native stereochemical configuration. Finally, we present evidence that shows that the class I MTs in marine crustacea are involved in the sequestration of elevated levels of heavy-metal ions. These observations strongly suggest that the different forms of MT have different biological functions.     

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.

     

Behavioral mechanisms influencing molt frequency in the American lobster,Homarus americanus Milne Edwards

The dominance relationship established among communally held juvenile lobsters (Homarusamericanus Milne Edwards) results in an increased time between molts of subordinates, slowing their growth. The experiments reported here elucidated some of the mechanisms of this behaviorally mediated change in growth rate. Rearing the lobsters in pairs on either side of a partition allowing chemical or visual communication showed that neither were sufficient to cause the decrease in molt rate. Removal of the claws reduced the extent of the molt delay and altered, but did not prevent, the development of the dominance relationship. The lobsters must be paired before proecdysis begins for the molt delay to occur. Most of the delay can be attributed to a lengthening of the D_o phase of proecdysis. Observations on the feeding behavior of pairs separated and together at feeding time showed the subordinates did not feed as readily or as rapidly as dominants. The slower growth rate of the subordinate may be due to a lower food intake.     

Production and characterization of recombinant vitellogenesis-inhibiting hormone from the American lobster Homarus americanus

Recombinant peptides related to vitellogenesis-inhibiting hormone (VIH) of the American lobster Homarus americanus were expressed in bacterial cells, and then purified after being allowed to refold. Biological activities of the recombinant VIHs having an amidated C-terminus (rHoa-VIH-amide) and a free carboxyl-terminus (rHoa-VIH-OH) were examined using an ovarian fragment incubation system derived from the kuruma prawn, Marsupenaeus japonicus. The rHoa-VIH-amide significantly reduced vitellogenin mRNA levels in the ovary, while rHoa-VIH-OH had no effect. This is the first report that describes the production of a crustacean VIH having biological activity and the importance of the C-terminal amidation for its vitellogenesis-inhibiting activity.     

Mitotic and meiotic chromosomes of the American lobster Homarus americanus (Nephropidae, Decapoda)

The chromosomes of H. americanus have been characterised by C-banding, fluorochrome banding and restriction endonuclease banding. Thanks to these techniques, it has been possible to identify mitotic and meiotic figures clearly and to study the distribution and structure of heterochromatic regions. Moreover, we have identified small supernumerary chromosomes, variable in number and often asynaptic in first meiotic metaphase.     

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.     

Short- and long-term dietary effects on disease and mortality in American lobster Homarus americanus

The American lobster Homarus americanus fishery is heavily dependent on the use of fish as bait to entice lobsters into traps. There is concern that this food supplementation is nutritionally insufficient for lobsters, but previous experiments reported conflicting results. We conducted a long-term feeding experiment in which 1 yr old American lobsters were fed one of 7 diets for a period of 352 d, a time that allowed the lobsters to molt thrice. The diets consisted of fresh frozen herring, a 'wild' diet (rock crab, mussel, and Spirulina algae), a formulated artificial diet for shrimp, paired combinations of these 3 diets or a diet formulated at the New England Aquarium (Artemia, fish and krill meal, Spirulina algae, soy lecithin, vitamins and minerals). The lobsters fed the diet of 100% fish had higher initial molting rates, but within the period of this experiment all either contracted shell disease or died. Mixed diets resulted in higher survival and a lower probability of mortality. This research demonstrated a critical time component to diet studies in lobsters. Short- and long-term impacts of diet differ. In the long term, continual high consumption rates of fish by the lobsters promote poor health in all lobsters, not just those of market size. The use of fish as bait may make lobsters more susceptible to the stress associated with environmental fluctuation, thereby leading to increased disease and mortality. This nutritional stress can be used to develop a laboratory model of shell disease in American lobsters.     

Pleistocene glaciation events shape genetic structure across the range of the American lobster, Homarus americanus

A north/south discontinuity along the northeastern coast of North America in the genetic structure of the American lobster ( Homarus americanus ) was detected using a suite of 13 microsatellite loci assessed using spatial analyses. Population genetic data laid over existing data on physiographic changes and sea-surface temperatures were used to reconstruct the Pleistocene distribution of this species. A postglacial northern-edge colonization model best explains the relative genetic homogeneity of the northern region compared to the southern region centred in the Gulf of Maine. Population genetic analyses identified significant structure (range of standardized theta 0–0.02) but no significant evidence for isolation by distance. The novel application of spatial genetic analyses to a marine species allowed us to interpret these results by providing a greater insight into the evolutionary factors responsible for shaping the genetic structure of this species throughout is natural range.     

The complete mitochondrial genome of the American lobster, Homarus americanus (Crustacea, Decapoda)

Although relatively a large number of the complete mitochondrial genome sequences have been determined from various decapod species (29 mtDNA sequences reported so far), the information for the infraorder Astacidea (including lobsters, crayfishes, and their relatives) is very limited and represented by only one complete sequence from the Australian freshwater crayfish species Cherax destructor. In this study, we determined the complete mitochondrial DNA sequence of Homarus americanus, the first representative of the family Nephropidae to be fully characterized. Comparison of the gene arrangement reveals that H. americanus mtDNA is identical to those of other pancrustacean species but differs from the other astacidean species (C. destructor). Based on these data, it can be assumed that an idiosyncratic gene order discovered in C. destructor mtDNA may be secondarily acquired from the ancestral lineage of the Astacidea.