Identifying potential prey of the pelagic larvae of the spiny lobster Jasus edwardsii using signature lipids

Signature lipid analyses were used to identify the natural prey of the pelagic phyllosoma larvae of the spiny lobster Jasus edwardsii. Lipid class, fatty acid and sterol composition were determined for associated potential prey items and for phyllosomes captured between 50 and 90 km offshore from the northeastern coast of New Zealand. Phospholipid was the dominant lipid class in all potential prey items and the transparent phyllosomes. The levels of other lipid classes varied between potential prey items, with the next most abundant classes being triacylglycerols, free fatty acids and sterols. A limited number of the potential prey items also contained wax ester. Major fatty acids in all potential prey items were generally 22:6ω3 (docosahexaenoic acid), 16:0, 18:1ω9c, and 20:5ω3 (eicosapentaenoic acid). Multivariate analyses of fatty acid and sterol content of the samples grouped the phyllosoma samples together regardless of their developmental stage or their collection location. However, the phyllosomes were not associated with any of the general groupings of pelagic ascidians, amphipods, chaetognaths, pteropods, euphausiids, fish, copepods or particulate matter that were formed by the statistical analyses. Although the sterol profiles of the potential prey items showed considerable variation, the phyllosomes contained predominately cholesterol, suggesting that other dietary sterols are converted to cholesterol or metabolised by the phyllosomes. Therefore, the use of sterols for tracing the prey of J. edwardsii phyllosoma appears to be limited. Our results suggest that phyllosomes are opportunistic predators that feed on a variety of prey and are preferentially retaining specific diet-derived fatty acids.     

Proximate control of diel vertical migration in Phyllosoma larvae of the Caribbean spiny lobster Panulirus argus

Phyllosoma larvae of the spiny lobster Panulirus argus undergo diel vertical migration (DVM), in which they are at depth during the day and nearer the surface at night. This study determined the visual spectral sensitivity of Stage I larvae and investigated whether light plays a proximate role in DVM as an exogenous cue and as an entrainment cue for an endogenous rhythm in vertical migration. Under constant conditions, larvae have a circadian rhythm (24.5-h period) in vertical swimming that resulted in a twilight DVM pattern. The behavioral response spectrum and electroretinogram recording indicated two photoreceptor spectral classes with maxima at 360 and 486 nm. When stimulated in an apparatus that simulated the underwater angular light distribution, dark-adapted larvae showed only positive phototaxis, with a threshold intensity of 1.8 × 10(13) photons m(-2) s(-1) (3.0 × 10(-5) μmoles photons m(-2) s(-1)). They have an avoidance response to predator shadows in which they descend upon sudden decreases in light intensity of more than 69%. When stimulated with relative rates of decrease in light intensity as occur at sunset they ascended, whereas they descended upon relative rates of light intensity increase as occur at sunrise. Thus, the DVM pattern is controlled by both an endogenous circadian rhythm in swimming and behavioral responses to light at sunrise and sunset.     

The ontogeny of physiological response to temperature in early stage spiny lobster (Jasus edwardsii) larvae

The physiological response to temperature, in terms of oxygen consumption, nitrogen excretion and feed intake was examined in Jasus edwardsii larvae at mid-stages I-III. From stage I to stage III, the mass-specific oxygen consumption increased in a sigmoid pattern over the temperature range of 10-22 degrees C. The Q(10) value declined significantly from 14-18 to 18-22 degrees C range, indicating a reduced temperature dependence of larval metabolism at higher temperatures. At all stages, feed intake increased with increasing temperature but reached a plateau at the higher temperatures for stages I and II larvae. In contrast, nitrogen excretion increased linearly over this temperature range for all larval stages. Therefore, higher temperatures ( approximately 22 degrees C) may cause an energetic imbalance and reduce growth potential in early stage larvae. While the convection requirement index (quotient of feed intake and oxygen consumption) indicated an equivalent metabolic feeding efficiency from 14 to 22 degrees C, a consistent decline of the O/N ratio above 16-18 degrees C from stage I to stage III suggested that exposure to elevated temperatures may result in an increase in the amount of protein being diverted from growth to catabolic processes. Based on these results, a temperature of 18 degrees C is recommended for the culture of early stage J. edwardsii larvae.     

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.     

Hydrogen ion titration and amino acid analysis of hemocyanin from the spiny lobster Jasus edwardsii.

Potentiometric and spectrophotometric titrations, isoelectric focusing and amino acid analyses, have been made on the hemocyanin from Jasus edwardsii. Counts of acidic neutral and alkaline groups were made from the titrations, enabling comparisons to be made with the amino acid analysis. Thermodynamic analysis of the data indicated that changes in the native protein structure took place at pH 4.0, 8.4 and 10.7. These observations are discussed in terms of dissociation, shifts in pK and conformational changes in the protein.     

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.     

HeLa cell identification by analysis of ribosomal DNA segment patterns generated by endonuclease restriction.

Restriction endonuclease analysis of HeLa cells and cells in which origins have been questioned provides evidence in favor of a HeLa cell origin for the questioned cells. Digestion of cellular human DNA reveals a variable ribosomal DNA (rDNA) fragment that is present in up to four discrete sizes. Cell lines of known and suspected HeLa origin contain only two size variants. This pattern of variability serves to distinguish HeLa-derived cells from others. Despite repeated passage and divergence of the HeLa phenotype and karyotype, the restriction pattern is remarkably constant.     

Organization of the stomatogastric ganglion of the spiny lobster

Summary Three direct synaptic connections occur between neurons in the gastric and pyloric systems of the stomatogastric ganglion ofPanulirus interruptus. Two synapses are inhibitory, and one is electrical. This electrical synapse is both excitatory and inhibitory at different times. These synapses, and others within each system, let the two systems interact under some conditions. The synapses also form multisynaptic pathways which modulate the firing of many neurons in both systems. The consequences of these multisynaptic pathways are described and discussed.I thank Allen I. Selverston, Karen Sigvardt, Eve Marder, David Russell and Mary Chamberlin for criticizing a draft of this paper, Forrest Gompf and Doug Tissdale for technical support, and Nina Pollack and Betty Jorgensen for laboratory assistance. The research was supported by USPHS grant NS-12295 to BM and USPHS grant NS-09322 to Alien I. Selverston. BM was a USPHS NIH Postdoctoral Fellow in A.I. Selverston's laboratory during part of this research and is now a Research Fellow of the Alfred P. Sloan Foundation.     

Threshold determination for olfactory receptors of the spiny lobster

The shallow water caridean shrimps Heptacarpus pictus and H. paludicola are polymorphic in colour pattern. Populations of these species collected over colour‐variable substrates showed the greatest degree of coloration in terms of the proportion of individuals displaying a distinct colour pattern. The frequencies of H. Pictus colour morphs varied significantly between most sampling periods. Apostatic selection by fish predators is suggested as one hypothesis which could explain these changes in morph frequency.

Experiments with Heptacarpus pictus on rapid colour change showed that, although some pigment migration did occur in the chromatosomes studied, the macroscopic appearance of the colour patterns was not altered when shrimps were shifted from black to white backgrounds or vice versa. The results of background choice experiments with H. pictus suggest that these shrimp do not seek out colour backgrounds that would seem to be a matching background in a concealing coloration. This behaviour is consistent with the morphology of the colour patterns which appears to be a disruptive coloration rather than a concealing coloration that closely matches a particular substrate.     

Species identification of five penaeid shrimps using PCR-RFLP and SSCP analyses of 16S ribosomal DNA

DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCRRFLP of 16S rDNA(560) whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S rDNA(560). Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S rDNA(560) of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S rDNA(312) was as effective as that of 16S rDNA(560). Differentiation of all shrimp species were successfully carried out by SSCP analysis.     

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.     

Cold-resistant changes in heartbeat of the Japanese spiny lobster

Heartbeat in Panulirus japonicus acclimated to 20°C is often augmented during cooling to 15^oC. Augmented contractions of the heart coincided with increasing amplitude of electrocardiogram. In cold saline, a pericardial hormone serotonin (10⁻⁷ M) increased both the amplitude and duration of the heartbeat while another hormone octopamine (10⁻⁶ M) slightly relieved the cold depression of heart rate despite a smaller increase in beat amplitude. In contrast, the application of the cold saline containing F1 (a FMRFamide-related peptide of pericardial hormones, 10⁻⁹ M) maintained the rate and amplitude of the heartbeat around the control level during cold exposure. This suggests that in the presence of F1, the lobster heart becomes cold resistant clearly. We previously reported that the pericardial organs of spiny lobsters are activated by a small fall in body temperature. The ligamental nerves, extensions of the pericardial organs, terminate in the heart beside the ostia and their ends remain in the isolated hearts. Therefore, the ligamental nerve ends might release their hormones into the ventricle with the fall in temperature even in the isolated hearts.     

Rapid identification of Phytophthora ramorum using PCR-SSCP analysis of ribosomal DNA ITS-1

AIMS: The primary objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis can be used for rapid identification of Phytophthora ramorum, an important quarantine plant pathogen worldwide, and to further assess the potential of the SSCP technique as a taxonomic tool for the genus Phytophthora. METHODS AND RESULTS: SSCP of ribosomal DNA internal transcribed spacer 1 was characterized for 12 isolates of P. ramorum, using a recently reported protocol. The SSCP patterns of this species then were compared with those of 18 closely related Phytophthora species. Phytophthora ramorum had a unique pattern and was easily distinguished from genetically, morphologically and ecologically close relatives. CONCLUSION: An immediate benefit of this study is provision of a highly effective and efficient identification tool for P. ramorum in the quarantine process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study also provides additional evidence demonstrating that the SSCP is an ideal DNA marker for species differentiation within the genus Phytophthora.     

Species delimitation in the African Armillaria complex by analysis of the ribosomal DNA spacers

Thirteen Armillaria isolates, collected from various geographical areas in tropical Africa and previously characterized by cultural morphology, pairing tests and isozyme analysis, were evaluated using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). DNA regions corresponding to the intergenic spacer (IGS) and internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The IGS amplification products were about 875 bp long and uniform in length among the isolates. The amplified-ITS region showed two different lengths corresponding to two groups. The first group included the isolates believed to belong to A. mellea ssp. africana and two Kenyan isolates (K11 and K12) belonging to a yet unnamed biological species. The second group included isolates identified as A. heimii and a Tanzanian isolate (T7). Each length variant of the ITS showed distinct RFLP banding patterns. Digestion with EcoRI confirmed the two polymorphic groups while the endonucleases AluI and NdeII discriminated the A. mellea isolates from the Kenyan isolates K11 and K12. In addition, the latter enzyme showed a slight dissimilarity between the A. heimii isolates from Western and Eastern Africa (C1 and Z1). Digestion with HinfI cleaved the isolates of A. heimii into two sub-groups corresponding to the heterothallic and homothallic forms. This endonuclease also indicated that the isolate T7, originating from Tanzania, was clearly similar to the heterothallic species A. heimii. Data presented support the maintenance of three distinct species of Armillaria in tropical Africa with A. heimii as a variable species, the isolates of which were separated in accordance with their sexual system. The results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for the ecological studies of Armillaria species.     

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.     

A late-stage phyllosoma larva of the spiny lobster Panulirus echinatus Smith, 1869 (Crustacea: Palinuridae) identified by DNA analysis

A late-stage phyllosoma larva of the genus Panulirus was caughtin the central Atlantic in October 2000. Nucleotide sequenceanalysis of mitochondrial 16S rDNA identified this larva asPanulirus echinatus, which has been undescribed to date. Morphologicalexamination indicated that this eighth stage larva belongedto phyllosoma species group 2, distinguishable from the otherAtlantic species of the genus but closely related to an Indo-Pacificspecies, P. penicillatus, which is in accordance with the resultsof a molecular phylogenetic analysis.