Specificity of the Induced Bactericidin of the West Indian Spiny Lobster, Panulirus argus

To investigate the specificity of an inducible hemolymph bactericidin, West Indian spiny lobsters were immunized with a variety of antigens. Bactericidal assays were conducted with strain EMB-1, a gram-negative bacillus isolated from normal lobster intestine. Animals given saline solution with or without 0.3% Formalin exhibited only a slight response similar to sham controls. Injections of type 2 pneumococci or bovine albumin produced slightly higher responses. These were much less pronounced than the homologous response to EMB-1. Formalin exhibited an adjuvant-like effect with EMB-1.     

The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny Lobster Panulirus argus

The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (K_i = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.     

Molecular Species Identification of Spiny Lobster Phyllosoma Larvae of the Genus Panulirus from the Northwestern Pacific

To identify lobster phyllosoma larvae of the genus Panulirus occurring in waters adjacent to Japan, genetic variation within and between 10 Indo-Pacific lobster species was investigated using restriction fragment length polymorphism (RFLP) analysis for the 1300-base pair mitochondrial cytochrome oxidase I (COI) gene. RFLP analysis using two endonucleases (AluI and TaqI) enabled discrimination of all species, including the P. longipes complex. The diagnostic DNA markers, supplemented with nucleotide sequence analysis, were applied to 44 mid- to late-stage phyllosoma larvae (7.4 to 27.7 mm in body length) collected in the northwestern Pacific. These samples were unexpectedly variable in species composition, comprising P. japonicus (n = 16), P. longipes bispinosus (21), P. longipes longipes (1), P. “aka” (1), and P. penicillatus (5). Comparison of larval size at similar stages revealed that P. l. bispinosus larvae were significantly larger than P. japonicus.     

Lobster (Panulirus argus) hepatopancreatic trypsin isoforms and their digestion efficiency

It is well known that crustaceans exhibit several isoforms of trypsin in their digestive system. Although the number of known crustacean trypsin isoforms continues increasing, especially those derived from cDNA sequences, the role of particular isoenzymes in digestion remains unknown. Among invertebrates, significant advances in the understanding of the role of multiple trypsins have been made only in insects. Since it has been demonstrated that trypsin isoenzyme patterns (phenotypes) in lobster differ in digestion efficiency, we used this crustacean as a model for assessing the biochemical basis of such differences. We demonstrated that the trypsin isoform known to be present in all individuals of Panulirus argus has a high catalytic efficiency (k(cat)/K(m) ) and is the most reactive toward native proteinaceous substrates, whereas one of the isoforms present in less efficient individuals has a lower k(cat) and a lower k(cat)/K(m), and it is less competent at digesting native proteins. A fundamental question in biology is how genetic differences produce different physiological performances. This work is the first to demonstrate that trypsin phenotypic variation in crustacean protein digestion relies on the biochemical properties of the different isoforms. Results are relevant for understanding trypsin polymorphism and protein digestion in lobster.     

Effect of Copper on Morphology, Weight, and Chromosomal Aberrations in the Spiny Lobster, Panulirus homarus (Linnaeus, 1758)

Spiny lobster Panulirus homarus which had been exposed to cupric ion at 9.55 and 19.1?μg/l for 28?days was examined for sub-lethal effects including morphology, wet weight, and induced genotoxic effect on the chromosome. Following cupric exposure, the color of lobster P. homarus changed from yellowish-brown to greenish black in the hepatopancreas, changed from normal creamy white to yellowish white in the muscle, and changed to greenish black in the gill. A significant change in the percentage of wet weight of muscle (28.70?±?0.41-23.47?±?0.45), hepatopancreas (4.03?±?0.12-2.63?±?0.17), and gills (3.63?±?0.45-3.87?±?0.12) were observed in the copper-treated lobsters. The diploid number of chromosomes of P. homarus was over 200 metaphases from ten lobsters, as 2n?=?58, and consisted of 16 acrocentric, seven metacentric, and six sub-metacentric chromosomes. The lobsters exposed to cupric ion at 9.55 and 19.1?μg/l showed different types of chromosomal aberrations such as centromeric gaps, chromatid breaks, centromeric fusion, stickiness, ring chromosomes, and acrocentric association region. The frequency of aberrations increased with duration of exposure. In conclusion, it was suggested that cupric ion interacts with the spindle formation and consequently distorts the normal karyomorphology, indicating cytogenetic effect on lobster.     

Primary culture of hemocytes from the Caribbean spiny lobster, Panulirus argus, and their susceptibility to Panulirus argus Virus 1 (PaV1)

Primary cultures of hemocytes from the Caribbean spiny lobster Panulirus argus were developed for studies on the in vitro propagation of Panulirus argus Virus 1 (PaV1). A modified Leibovitz L-15 medium supported the best survival of hemocytes in in vitro primary cultures. However, degradation of the cultures occurred rapidly in the presence of granulocytes. A Percoll step gradient was used to separate hemocytes into three subpopulations enriched in hyalinocytes, semigranulocytes, and granulocytes, respectively. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability ( approximately 80%) after 18 days incubation compared with granulocytes, which degraded over 2-3 days. Susceptibility of the cell types was investigated in challenge studies with PaV1. Hyalinocytes and semigranulocytes were susceptible to PaV1. Cytopathic effects (CPE) were observed as early as 12h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates present in inoculated cultures. Cell lysis was noticeable within 24h of infection. The presence of virus within cells was further confirmed by in situ hybridization using a specific DNA probe. The probe gave a unique staining pattern to cells infected with PaV1 24-h post-inoculation. Cells in the control treatment were intact and negative to hybridization. This assay was further applied to the quantification of infectious virus in hemolymph using a 50% tissue culture infectious dose assay (TCID(50)) based on CPE. These tools will now allow the quantification of PaV1 using established culture-based methods.     

Presence of Panulirus argus virus 1 (PaV1) in juvenile spiny lobsters Panulirus argus from the Caribbean coast of Mexico

Macroscopic evidence, histological sections, transmission electron microscopy (TEM) evaluation, and PCR analyses of 25 apparently diseased juvenile spiny lobsters Panulirus argus from the reef lagoon of Puerto Morelos, Mexico, showed the presence of Panulirus argus Virus 1 (PaV1). Cowdry Type A intranuclear viral inclusions were observed in histological analyses, icosahedral viral particles were observed by TEM, and PCR using specific primers for PaV1 amplified a fragment of 499 bp. This is the first report of PaV1 infecting P. argus outside the Florida Keys, USA.     

Isolation and characterization of the mitochondrial DNA from the florida spiny lobster, Panulirus argus

1. Mitochondria and mitochondrial DNA were isolated from the digestive gland of Panulirus argus. 2. The mitochondrial DNA has an average contour length of 5.13 microns which corresponds to a molecular weight of 10.10 X 10(6) daltons. 3. The molecular weight based on agarose gel electrophoresis of restricted individual DNA samples ranges from 10.04 to 10.4 X 10(6) daltons. 4. Restriction endonuclease analysis with Bam H1 and Eco R1 demonstrate variation in nucleotide sequence between individual lobsters.     

Ectonucleotidase Activities Associated with the Olfactory Organ of the Spiny Lobster

The olfactory system of the Florida spiny lobster, Panulirus argus, has olfactory receptors that are excited by the purine nucleotides AMP, ADP, and ATP. These receptors reside on chemosensory neurons that are contained within aesthetasc sensilla on the lateral filaments of the antennules. Also associated with the lobster's olfactory system are ectonucleotidase activities that dephosphorylate excitatory nucleotides, resulting in the production of the nonstimulatory nucleoside adenosine. Our studies of the 5'-ectonucleotidase, ecto-ADPase, and ecto-ATPase activities of this olfactory system showed that each activity was characterized by Michaelis-Menten kinetics; Michaelis constants ranged from 6.9 to 33.5 microM, and maximum velocities ranged from 2.5 to 28.8 fmol/sensillum/s. Evidence that AMP dephosphorylation may serve as an inactivation process was shown by the close correlation between the kinetics of 5'-ectonucleotidase activity and the periodicity of olfactory sampling. Decreased magnesium ion concentration or increased calcium ion concentration resulted in increased ecto-ATPase activity; this activity was insensitive to vanadate ion. Ectonucleotidase activities may have multiple effects on the detection of exogenous nucleotides by a chemosensory system. These effects can be either direct, such as the conversion of an odorant to an inactive compound, or indirect, such as the conversion of an odorant to another compound that can activate or inhibit either receptors or enzymes associated with the system.     

Polymorphic tetranucleotide microsatellite markers in the Caribbean spiny lobster,Panulirus argus

Ten tetranucleotide microsatellite loci are described for the Caribbean spiny lobster Panulirus argus. Loci were polymorphic (4–15 alleles per locus) and exhibited high levels of expected (0.553–0.921) and observed heterozygosity (0.469–0.906) from samples caught off Belize and Puerto Rico coasts. No significant departure from Hardy–Weinberg equilibrium conditions were observed for any locus. All microsatellite loci should be useful for assessing population discrimination for this valuable marine animal currently subjected to excessive fishing efforts.     

Isolation, ultrastructure, and partial characterization of collagen from the perineurium of the Florida lobster, Panulirus argus.

Highly concentrated extracellular filaments in the perineurium of the Florida spiny lobster, Panulirus argus, were isolated using ultracentrifugation and linear sucrose gradients. The pellet obtained was highly enriched for the filaments as observed by transmission electron microscopy. Fibril diameter and axial periodicity measurements were obtained from filaments positively and negatively stained with uranyl acetate. A period between 14.0 and 25.0 nm and an average fibril diameter of 15.0 nm were observed. The filaments proved resistant to solubilization by most conventional agents and by several collagenases. NaOH (0.1 M at 100 degrees C) safely dissolved the filaments for measurements of protein content by the Lowry method and carbohydrate content with anthrone reagent. These tests revealed a protein content of approximately 84% and a high carbohydrate content of approximately 15%. Polyacrylamide electrophoresis of an acid-pepsin filament extract revealed a highly concentrated band (approximately 100,000) corresponding to the alpha-1 and alpha-2 bands of vertebrate type I collagen. Wide angle X-ray diffraction yielded meridional reflections that confirmed the filaments as collagen when compared with mammalian collagen X-ray diffraction. The amino acid composition was determined with a computer-assisted Beckman amino acid analyzer, which showed a glycine content of 279 residues/1000. Hydroxylysine and hydroxyproline were present in lower concentrations than expected.     

Perception of odor mixtures by the spiny lobster Panulirus argus

We used spiny lobsters (Panulirus argus) in a discriminationlearning procedure with aversive conditioning to examine theirbehavioral discrimination of adenosine-5'-monophosphate (AMP),betaine, L-cysteine and their binary mixtures. Our results showthat spiny lobsters can clearly discriminate among binary mixturesand their components. Lobsters aversively conditioned to avoidresponding to a binary mixture continued to respond to thatmixture's components, and lobsters that were aversively conditionedto avoid responding to a compound tended to continue to respondto binary mixtures containing that compound. Thus, responsesof conditioned lobsters to binary mixtures were not usuallyintermediate between the responses to the mixtures' components,which might be expected for response-matched compounds. Thisresult might arise from any of several factors. First, it mightresult from mixture interactions in the peripheral olfactorysystem, if the responses of olfactory receptor neurons to onecomponent of a binary mixture were suppressed by the other component,making the response to the mixture more similar to the suppressingcomponent. Electrophysiological data from a population of 50singly-recorded olfactory receptor neurons (Daniel and Derby,1994) do not consistently support this idea. A second possiblereason for the behavioral response to a binary mixture not beingintermediate between the responses to its components involveshigher order processing, such as mixture interactions generatedin olfactory interneurons in the CNS (which is known to occur:Derby et al., 1985; Ache, 1989), configural learning or associativeprocessing.     

Processing of antennular input in the brain of the spiny lobster, Panulirus argus

Neurons in the olfactory deutocerebrum of the spiny lobster, Panulirus argus, were recorded intracellularly and filled with biocytin. Recorded neurons arborized in the olfactory lobe (OL), a glomerular neuropil innervated by olfactory and some presumptive mechanosensory antennular afferents. The neurons responded to chemosensory input from the lateral antennular flagellum bearing the olfactory sensilla but not the medial flagellum bearing many non-olfactory chemosensory sensilla. Many neurons received additional mechanosensory input. Thus the OL integrates specifically olfactory with mechanosensory input. OL neurons had multiglomerular arborizations restricted to one or two of the three horizontal layers of the columnar glomeruli. OL local interneurons comprised core neurons with tree-like neurites and terminals in the base of the glomeruli and rim neurons with neurites surrounding the OL and terminals in the cap/subcap. The somata of OL local interneurons lay in the medial soma cluster (100000 somata). OL projection neurons arborized in the base of the glomeruli and ascended via the olfactory glomerular tract to the lateral protocerebrum. A parallel projection pathway is constituted by projection neurons of the accessory lobe, a glomerular neuropil without afferent innervation but intimate links to the OL. The projection neuron somata constituted the lateral soma cluster (200000 somata).Abbreviations AC anterior cluster (cluster 6,7) - AL accessory lobe - aMC anterior subcluster of medial cluster (cluster 9) - A _lNv main antenna I (antennular) nerve - A _lNM antenna I (antennular) motor nerve - A _llNv main antenna II (antennal) nerve - CB central body - CL central layer of accessory lobe - DC deutocerebral commissure - DCN deutocerebral commissure neuropil - dDUMC dorsal subcluster of dorsal unpaired median cluster (cluster 17) - dMC dorsal subcluster of medial cluster (cluster 11) - dVPALC dorsal subcluster of ventral paired anterolateral cluster (cluster 8) G glomerulus - IDUMC lateral subcluster of dorsal unpaired median cluster (cluster 16) - LC lateral cluster (cluster 10) - LF lateral flagellum of antenna I (antennule) - LL lateral layer of accessory lobe - MF medial flagellum of antenna I (antennule) - ML medial layer of accessory lobe - MPN anterior and posterior median protocerebral neuropils - OGT olfactory globular tract - OGTN olfactory globular tract neuropil - OL olfactory lobe - OLALT olfactory lobe-accessory lobe tract - PB protocerebral bridge - pMC posterior subcluster of medial cluster (cluster 9) - PT protocerebral tract - TNv tegumentary nerve - VPMC ventral paired medial cluster (cluster 12) - VUMC ventral unpaired medial cluster (cluster 13) - vVPALC ventral subcluster of ventral paired anterolateral cluster (cluster 8) - ASW artificial sea water - M3 mixture 3 - PRO L-proline - TM TetraMarin extract     

Oceanographic Currents and Local Ecological Knowledge Indicate,and Genetics Does Not Refute,a Contemporary Pattern of Larval Dispersal for The Ornate Spiny Lobster,Panulirus ornatus in the South-East Asian Archipelago

Here we utilize a combination of genetic data, oceanographic data, and local ecological knowledge to assess connectivity patterns of the ornate spiny lobster Panulirus ornatus (Fabricius, 1798) in the South-East Asian archipelago from Vietnam to Australia. Partial mitochondrial DNA control region and 10 polymorphic microsatellites did not detect genetic structure of 216 wild P. ornatus samples from Australia, Indonesia and Vietnam. Analyses show no evidence for genetic differentiation among populations (mtDNA control region sequences Φ_ST = -0.008; microsatellite loci F_ST = 0.003). A lack of evidence for regional or localized mtDNA haplotype clusters, or geographic clusters of microsatellite genotypes, reveals a pattern of high gene flow in P. ornatus throughout the South-East Asian Archipelago. This lack of genetic structure may be due to the oceanography-driven connectivity of the pelagic lobster larvae between spawning grounds in Papua New Guinea, the Philippines and, possibly, Indonesia. The connectivity cycle necessitates three generations. The lack of genetic structure of P. ornatus population in the South-East Asian archipelago has important implications for the sustainable management of this lobster in that the species within the region needs to be managed as one genetic stock.     

Mechanosensory neurons with bend- and osmo-sensitivity in mouthpart setae from the spiny lobster Panulirus argus

The mouthparts of the spiny lobster Panulirus argus hold primarily two types of setae--simple setae and cuspidate setae. Mechanosensory neurons from these setae were examined by electrophysiological recordings. The population of simple setae contained two types of mechanosensory neurons: displacement-sensitive neurons, which responded to deflection at the setal base; and bend-sensitive neurons, which responded to bending of the setal shaft. Displacement-sensitive neurons, in general, responded phasically and only during actual displacement. Typically, their response changed with alteration of the direction, amplitude, and velocity/acceleration of the mechanical stimulus. Bend-sensitive neurons, in general, responded phaso-tonically and carried information on the direction and region of bending. This is the first experimental demonstration of bend sensitivity for arthropod setae. Cuspidate setae contain highly sensitive mechanosensory neurons; however, due to the rigid nature of these setae, whether they were bend sensitive or displacement sensitive could not be determined, and they were thus called "tactile neurons." Bend-sensitive neurons, but not displacement-sensitive neurons or tactile neurons, showed graded responses to changes in osmolarity. The osmosensitivity of these neurons could mediate behavioral responses to changes in the osmolarity of seawater or food.     

Phenoloxidase activity in the hemolymph of the spiny lobster Panulirus argus

The prophenoloxidase activating system plays a major role in the defense mechanism of arthropods. In the present study, the phenoloxidase activity and its location in the hemolymph of the spiny lobster Panulirus argus is presented. Phenoloxidase activity was observed in the hemocyte lysate supernatant (HLS) and plasma after their incubation with trypsin. Higher amounts of trypsin were required to activate the HLS prophenoloxidase, due to the presence of a trypsin inhibitor in this fraction. Activation of prophenoloxidase was found when HLS was incubated with calcium, with an optimal pH between 7.5 and 8. This spontaneous activity is due to the prophenoloxidase activating enzyme, a serine proteinase that activates the prophenoloxidase once calcium ions were available. SDS was able to induce phenoloxidase activity in plasma and hemocyte fractions. Prophenoloxidase from HLS occurs as an aggregate of 300kDa. Electrophoretic studies combining SDS-PAGE and native PAGE indicate that different proteins produced the phenoloxidase activity found in HLS and plasma. Thus, as in most crustaceans, Panulirus argus contains a prophenoloxidase activating system in its hemocyte, comprising at least the prophenoloxidase activating enzyme and the prophenoloxidase. Finally, it is suggested that phenoloxidase activity found in plasma is produced by hemocyanin.     

Loss of escape-related giant neurons in a spiny lobster, Panulirus argus

When attacked, many decapod crustaceans perform tailflips, which are triggered by a neural circuit that includes lateral giant interneurons, medial giant interneurons, and fast flexor motor giant neurons (MoGs). Slipper lobsters (Scyllaridae) lack these giant neurons, and it has been hypothesized that behavioral (e.g., digging) and morphological (e.g., flattening and armor) specializations in this group caused the loss of escape-related giant neurons. To test this hypothesis, we examined a species of spiny lobster, Panulirus argus. Spiny lobsters belong to the sister taxon of the scyllarids, but they have a more crayfish-like morphology than scyllarids and were predicted to have escape-related giant neurons. Ventral nerve cords of P. argus were examined using paraffin-embedded sections and cobalt backfills. We found no escape-related giant neurons and no large axon profiles in the dorsal region of the nerve cord of P. argus. Cobalt backfills showed one fewer fast flexor motor neuron than in species with MoGs and none of the fast flexor motor neurons show any of the anatomical specializations of MoGs. This suggests that all palinuran species lack this giant escape circuit, and that the loss of rapid escape behavior preceded, and may have driven, alternative predator avoidance and anti-predator strategies in palinurans.