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.     

Laboratory comparison of American lobster,Homarus americanus,foraging responses to invasive green crab,Carcinus maenas,and two traditional finfish baits

The American Lobster (Homarus americanus) is trapped primarily with finfish baits that are becoming more costly and difficult to acquire as stocks dwindle. Thus, alternative bait options should be considered. We made video recordings of lobster foraging responses in the laboratory to compare the attractiveness of the invasive green crab (Carcinus maenas) bait, two traditional finfish baits and an inert control. Solitary lobsters approached and handled all three biological baits with similar latencies and spent similar time in contact with the biological baits. In contrast, lobsters took longer to approach and handle, and spent significantly less time in contact with control bait. As green crabs were statistically indistinguishable in attractiveness from traditional baits in our laboratory setting, we suggest field trials should test this alternate bait in circumstances more directly relevant to the fishery.     

Quantifying factors influencing American lobster (Homarus americanus) predation on the invasive green crab (Carcinus maenas) in Newfoundland,Canada

The European green crab (Carcinus maenas) is a newly invasive species in Newfoundland, where it has likely been present for ≤15 years. The green crab has been found in stomach contents of American lobster (Homarus americanus) in New England and Nova Scotia, Canada, but predation on this species has not yet been quantified in Newfoundland. We conducted feeding experiments to determine whether lobsters from Newfoundland were as likely as those from Nova Scotia (which have coexisted with green crabs for >60 years) to recognize and prey upon this new species. We also performed experiments to determine whether green crabs reach a size refuge from predation and whether factors including starvation, availability of alternate food sources, or habitat complexity would influence the probability of lobster attacking or feeding on green crabs. In our trials, lobster origin had no significant effect on crab predation; lobsters, irrespective of origin, were more likely to consume small (<40 mm carapace width [CW]) and medium (40–65 mm CW) crabs than larger (>65 mm CW) ones. Nevertheless, even small lobsters (73–76 mm carapace length, 300 g) were able to kill and consume the largest green crabs (78 mm CW, 100 g). Green crabs were less likely to be attacked or eaten when an alternative food source was present, suggesting that the lobsters were preying on the crabs, rather than simply killing them in a dispute over territory. The addition of a shelter provided a refuge for the green crabs; however, the crabs were only able to avoid being injured or eaten if this shelter was structurally complex. The green crab is slowly spreading westward around the island of Newfoundland, and so its long‐term effects, interactions with other organisms, and contribution to the diet of Newfoundland lobsters remain to be seen.     

An historical overview of the Republic of Ireland's lobster (Homarus gammarus Linnaeus) fishery,with reference to European and North American (Homarus americanus Milne Edwards) lobster landings

Historical developments of the Republic of Ireland's Homarus gammarus fishery are reviewed and compared in the context of landings in Europe and the North American H. americanus fishery. H. gammarus fishing has provided essential income for over a century to many Irish coastal communities. There have been significant fluctuations (greater than 460%) in recorded landings over this time and the relative importance of H. gammarus fishing has changed with the establishment of ancillary crustacean fisheries. In 1994, H. gammarus landings reached an all-time high of 714 tonnes, with a landed value of IR £6.3 million; in 1999 landings were 597 tonnes valued at IR £6.15 million. Current Republic of Ireland lobster fishery regulations are: (a) minimum carapace length of 85 mm, (b) ban on landing lobsters that have been `V-notched' or have a mutilated tail fan, and (c) prohibition of capturing lobsters by SCUBA diving. Chronological details on recent legislation, regulations and landings relating to Ireland's lobster fishery sector are reviewed. In the last 40 years landings of H. gammarus in the Republic of Ireland exhibit signs of an overexploited stock characterised by a reduction in catch per unit effort and there has been a general decline of total landings for H. gammarus throughout Europe. This contrasts with North America, where many regulations govern the H. americanus fishery and landings appear to be in a healthier state.     

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.     

Circadian rhythms of heart rate in freely moving and restrained American lobsters, Homarus americanus

While circadian rhythms of locomotion have been reported in the American lobster, Homarus americanus, it is unclear whether heart rate is also modulated on a circadian basis. To address this issue, both heart rate and locomotor activity were continuously monitored in light-dark (LD) cycles and constant darkness (DD). Lobsters in running wheels exhibited significant nocturnal increases in locomotor activity and heart rates during LD, and these measures were significantly correlated. In DD, most lobsters exhibited persistent circadian rhythms of both locomotion and heart rate. When heart rate was monitored in restrained lobsters in LD and DD, most animals also demonstrated clear daily and circadian rhythms in heart rate. Overall, this is the first demonstration of circadian rhythms of heart rate in H. americanus, the expression of which does not appear to be dependent on the expression of locomotor activity.     

Circadian rhythms of heart rate in freely moving and restrained American lobsters,Homarus americanus

While circadian rhythms of locomotion have been reported in the American lobster, Homarus americanus, it is unclear whether heart rate is also modulated on a circadian basis. To address this issue, both heart rate and locomotor activity were continuously monitored in light-dark (LD) cycles and constant darkness (DD). Lobsters in running wheels exhibited significant nocturnal increases in locomotor activity and heart rates during LD, and these measures were significantly correlated. In DD, most lobsters exhibited persistent circadian rhythms of both locomotion and heart rate. When heart rate was monitored in restrained lobsters in LD and DD, most animals also demonstrated clear daily and circadian rhythms in heart rate. Overall, this is the first demonstration of circadian rhythms of heart rate in H. americanus, the expression of which does not appear to be dependent on the expression of locomotor activity.     

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female''s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George''s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.     

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.     

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.     

RAD genotyping reveals fine‐scale genetic structuring and provides powerful population assignment in a widely distributed marine species,the American lobster (Homarus americanus)

Deciphering genetic structure and inferring connectivity in marine species have been challenging due to weak genetic differentiation and limited resolution offered by traditional genotypic methods. The main goal of this study was to assess how a population genomics framework could help delineate the genetic structure of the American lobster (Homarus americanus) throughout much of the species’ range and increase the assignment success of individuals to their location of origin. We genotyped 10 156 filtered SNPs using RAD sequencing to delineate genetic structure and perform population assignment for 586 American lobsters collected in 17 locations distributed across a large portion of the species’ natural distribution range. Our results revealed the existence of a hierarchical genetic structure, first separating lobsters from the northern and southern part of the range (F_CT = 0.0011; P‐value = 0.0002) and then revealing a total of 11 genetically distinguishable populations (mean F_ST = 0.00185; CI: 0.0007–0.0021, P‐value < 0.0002), providing strong evidence for weak, albeit fine‐scale population structuring within each region. A resampling procedure showed that assignment success was highest with a subset of 3000 SNPs having the highest F_ST. Applying Anderson's (Molecular Ecology Resources, 2010, 10, 701) method to avoid ‘high‐grading bias’, 94.2% and 80.8% of individuals were correctly assigned to their region and location of origin, respectively. Lastly, we showed that assignment success was positively associated with sample size. These results demonstrate that using a large number of SNPs improves fine‐scale population structure delineation and population assignment success in a context of weak genetic structure. We discuss the implications of these findings for the conservation and management of highly connected marine species, particularly regarding the geographic scale of demographic independence.     

Development of EST‐SSRs from the Alpine Lady‐fern,Athyrium distentifolium

The utility of EST‐simple sequence repeats (EST‐SSRs) was evaluated in the fern Athyrium distentifolium. From 1152 frond cDNA clones, 165 microsatellites, including di‐, tri‐, tetra and penta‐nucleotide repeat motifs, were identified. Primer design was possible for 74 of the SSRs; subsequent screening of 10 loci on 186 individuals from six natural populations revealed between two and seven alleles per locus and expected heterozygosity (H_E) estimates ranging from 0.027 to 0.809. Eight of these loci were further examined for cross‐species and cross‐generic amplification in other Woodsiaceae species, and polymorphic products were detected. EST‐derived SSRs provide robust, informative and potentially transferable polymorphic markers suitable for biodiversity research.     

Neuromuscular synapses on the dactyl opener muscle of the lobster Homarus americanus

The crustacean dactyl opener neuromuscular system has been studied extensively as a model system that exhibits several forms of synaptic plasticity. We report the ultrastructural features of the synapses on dactyl opener of the lobster (Homarus americanus) as determined by examination of serial thin sections. Several innervation sites supplied by an inhibitory motoneuron have been observed without nearby excitatory innervation, indicating that excitatory and inhibitory inputs to the muscle are not always closely matched. The ultrastructural features of the lobster synapses are generally similar to those described previously for the homologous crayfish muscle, with one major distinction: few dense bars are seen at the presynaptic membranes of these lobster synapses. The majority of the lobster neuromuscular synapses lack dense bars altogether, and the mean number of dense bars per synapse is relatively low. In view of the finding that the physiology of the lobster dactyl opener synapses is similar to that reported for crayfish, these ultrastructural observations suggest that the structural complexity of the synapses may not be a critical factor determining synaptic plasticity.This work was supported by funds from the University of Virginia Research and Development Committee.     

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.     

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.     

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.     

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.     

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).