Identification of an antagonistic probiotic combination protecting ornate spiny lobster (Panulirus ornatus) larvae against Vibrio owensii infection

Vibrio owensii DY05 is a serious pathogen causing epizootics in the larviculture of ornate spiny lobster Panulirus ornatus. In the present study a multi-tiered probiotic screening strategy was used to identify a probiotic combination capable of protecting P. ornatus larvae (phyllosomas) from experimental V. owensii DY05 infection. From a pool of more than 500 marine bacterial isolates, 91 showed definitive in vitro antagonistic activity towards the pathogen. Antagonistic candidates were shortlisted based on phylogeny, strength of antagonistic activity, and isolate origin. Miniaturized assays used a green fluorescent protein labelled transconjugant of V. owensii DY05 to assess pathogen growth and biofilm formation in the presence of shortlisted candidates. This approach enabled rapid processing and selection of candidates to be tested in a phyllosoma infection model. When used in combination, strains Vibrio sp. PP05 and Pseudoalteromonas sp. PP107 significantly and reproducibly protected P. ornatus phyllosomas during vectored challenge with V. owensii DY05, with survival not differing significantly from unchallenged controls. The present study has shown the value of multispecies probiotic treatment and demonstrated that natural microbial communities associated with wild phyllosomas and zooplankton prey support antagonistic bacteria capable of in vivo suppression of a pathogen causing epizootics in phyllosoma culture systems.     

Behavioral thermoregulation in the spiny lobster Panulirus Argus (latreille)

Ten spiny lobsters (Panulirus argus) were allowed to thermoregulate individually for 3-day periods in an electronic thermoregulatory shuttlebox which allowed them to control water temperatures (and thereby their own body temperatures) by their movements. The range of preferred (voluntarily occupied) temperatures was 25–35°C (mean 29.9°C; mode 30.0°C; median 30.0°C; midpoint 30.0°C; S_k (skewness, Pearson's coefficient) –0.04; s.e.m. 0.19°C; S.D. 2.32°C). The final thermal preferendum (by the gravitation method) in this species is 30°C.     

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Glucose and sulfate conjugation of phenolic compounds by the spiny lobster (Panulirus argus)

Previous work has shown that the hepatopancreas of the spiny lobster (Panulirus argus) contains a mixed-function oxidase system capable of catalyzing the monooxygenation of polycyclic aromatic hydrocarbons to highly toxic products similar to those formed by mammalian tissues. Studies were designed to determine the ability of the spiny lobster to conjugate the phenolic compounds 4-methylumbelliferone, p-nitrophenol, beta-naphthol, and 3-hydroxybenzo[a]pyrene with endogenous molecules. The hepatopancreas contained UDP-glucose (UDPG) dependent glucosyltransferase, while no activity was detected when UDP-glucuronic acid was used as the cosubstrate. Atypical Michaelis-Menten kinetics result with varying concentrations of UDPG, indicating that multiple forms of glucosyltransferase may exist in this organ. The activity was localized in the microsomal fraction, exhibited a pH optimum at 8.0-8.5, and a temperature optimum of 30 degrees C. Sulfate conjugation was found only in the cytosolic fraction of the antennal gland and used adenosine 3'-phosphate 5'-phosphosulfate (PAPS) as the sulfate donor (Km(apparent) = 9.0 +/- 4.9 microM). Hepatopancreas cytosol inhibited sulfotransferase activity. The pH optimum of antennal gland sulfotransferase was a function of the substrate and ranged from 5.5 to 7.4. Analysis of spiny lobster urine 24 hr following exposure to 3-hydroxybenzo[a]pyrene demonstrated the ability of the lobster to form both the sulfate and glucoside conjugate in vivo.     

Serum lipoproteins in the spiny lobster, Panulirus interruptus.

1. Most of the lipids in the hemolymph of the spiny lobster, Panulirus interruptus, were associated with a high density lipoprotein (HDL3). The lipid of this lipoprotein was composed of phospholipid (88%), sterol (4%) and triglyceride (3%). 2. In animals fed 14C-labeled triglyceride radioactivity was not seen in the serum until 12 hr after feeding. Most of this serum radioactivity was associated with phosphatidyl choline. 3. Electron micrographs showed that negatively stained high density lipoproteins of the lobster had a polymorphic appearance.     

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.     

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.     

In the spiny lobster (Panulirus interruptus) the prophenoloxidase is located in plasma not in haemocytes

In the spiny lobster (Panulirus interruptus), unlike other crustaceans most of the prophenoloxidase (proPO) was detected in cell-free plasma (86.3%). In spite of its location, lobster proPO activating system has a similar activation mechanism to other crustacean proPO systems. Haemocyte lysate was able to activate the plasma proPO indicating location of the prophenoloxidase activating enzyme (PPAE) in haemocytes. Lobster haemocyte PPAE was isolated by affinity chromatography and its participation as activating enzyme was demonstrated. This enzyme is a serine-proteinase that transforms the inactive form (proPO) to an active one (phenoloxidase). The PPAE was also present in the cell-free supernatant of haemocytes previously incubated with Vibrio alginolyticus.     

Tripartite genetic subdivisions in the ornate shrew (Sorex ornatus)

We examined cytochrome b sequence variation in 251 ornate shrews (Sorex ornatus) from 20 localities distributed throughout their geographical range. Additionally, vagrant (S. vagrans) and montane (S. monticolus) shrews from four localities were used as outgroups. We found 24 haplotypes in ornate shrews from California (USA) and Baja California (Mexico) that differed by 1-31 substitutions in 392 bp of mitochondrial DNA (mtDNA) sequence. In a subset of individuals, we sequenced 699 bp of cytochrome b to better resolve the phylogeographic relationships of populations. The ornate shrew is phylogeographically structured into three haplotype clades representing southern, central and northern localities. Analysis of allozyme variation reveals a similar pattern of variation. Several other small California vertebrates have a similar tripartite pattern of genetic subdivision. We suggest that topographic barriers and expansion and contraction of wetland habitats in the central valley during Pleistocene glacial cycles account for these patterns of genetic variation. Remarkably, the northern ornate shrew clade is phylogenetically clustered with another species of shrew suggesting that it may be a unique lowland form of the vagrant shrew that evolved in parallel to their southern California counterparts.     

Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.     

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.     

Behavioral olfactory discrimination of mixtures in the spiny lobster (Panulirus argus) based on a habituation paradigm

A habituation paradigm was used to examine behavioral aspectsof discrimination of stimulus quality in olfaction in the spinylobster (Panulirus argus). Magnitude of search response to twoconcentrations of artificial mixtures of crab, oyster, mulletand shrimp and to artificial seawater was measured in five lobstersbefore and immediately after habituation to crab mixture. Habituationto crab mixture was accomplished through 2-min presentationsof 5 ml of alternating concentrations (0.05 and 0.5 mM) of crabmixture stimulus, repeated every 5 min for a total durationof at least 3 h. This procedure resulted in at least a 42% decreasein response to any mixture, but the decrease was greatest forcrab mixture, the habituating stimulus. A habituation index,measured as post-habituation response relative to pre-habituationresponse, was used to evaluate discrimination between crab andeach of the other three mixtures. The habituation index wassignificantly greater for crab (90%) than for oyster (49%) andfor mullet (47%) mixtures. The habituation index for shrimpmixture (65%) was intermediate to these three mixtures. Thus,shrimp mixture is perceived by lobsters as being more similarto crab mixture than is either oyster or mullet mixture. Thesepatterns of discrimination parallel those reported for associativelyconditioned lobsters (Fine-Levy et al., 1987, 1988) and fora population of olfactory receptor cells (Girardot and Derby,1988).     

Complete mitochondrial DNA sequence of the Japanese spiny lobster,Panulirus japonicus (Crustacea: Decapoda)

We determined the complete nucleotide sequence of the mitochondrial genome for a Japanese spiny lobster, Panulirus japonicus (Crustacea: Decapoda). The entire genome was amplified using long polymerase chain reaction, and the products were subsequently used as templates for direct sequencing using a primer-walking strategy. The genome (15,717 base pairs) contained the same 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) plus the putative control region as found in other arthropods, with the gene order identical to that of typical arthropods. Preliminary phylogenetic analyses of selected arthropods using concatenated amino acid sequences of the 13 protein-coding genes strongly supported monophyly of Decapoda species and confidently rejected "Macroura", a conventional taxon that shares an elongated abdominal body.     

Decadal variability in growth of the Caribbean spiny lobster Panulirus argus (Decapoda: Paniluridae) in Cuban waters

Annual von Bertalanffy growth parameters of the Caribbean spiny lobster (Panulirus argus) in Cuban waters were estimated from a long term study (40 years) by length-based methods ELEFAN and the new version of SLCA. Data of around 800 000 lobsters (with carapace length ranging 14 to 199mm) were randomly sampled in artificial shelters (a non selective fishing gear very common in the lobster fishery), through the field monitory program established for this species since 1963 in 14 localities of southwestern Cuban shelf. The software ELEFAN showed problems to converge in an optimal combination of the instantaneous growth coefficient (K) and the asymptotic length (Linfinity) of the von Bertalanffy equation, whereas the new SLCA software produced value estimates of K between 0.20 and 0.27 year(-1) and values of Linfinity between 177 and 190 mm carapace length, all within the range reported in the literature. The standardized anomalies of both parameters showed the presence of cycles along the analyzed time series. Decadal variability in growth parameters was revealed through the spectral analysis indicating cycles of 16 and 20 years for K and of 16 years for Linfinity. The incidence of some factors such as biomass and temperature that modulate growth in this crustacean was explored, using a nonlinear multiple regression model. These combined factors explained 33% and 69% of the variability of K and Linfinity respectively. The growth coefficient appeared to be maximum with annual mean sea surface temperature of 28. 1 degrees C and the largest Linfinity is reached at a annual men biomass level of 23,000 t. These results should be the basis to understand the Cuban lobster population dynamics.     

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.