John Benzie

Genetic mapping of the black tiger shrimp Penaeus monodon with amplified fragment length polymorphism

Abstract We report construction of an initial genetic linkage map for the black tiger shrimp, Penaeus monodon. Mapping was carried out using polymorphic markers derived from 23 Amplified Fragment Length Polymorphism (AFLP) primer pairs. These were analysed on three reference families of known pedigree. A total of 673 polymorphic AFLP loci that conformed to expected Mendelian segregation ratios were scored in three families, and these were used to construct separate male and female linkage maps for each family. AFLP markers that consisted of a segregating fragment of the same size, amplified with the same primer pair in two or more of the reference families, were considered to be common markers. 116 such common AFLP markers were used to construct a common linkage map across the three families. This linkage map has 20 linkage groups covering a total genetic distance of 1412 cM. Future directions for genetic mapping in P. monodon are discussed in light of these initial data.

Major genetic differences between crown-of-thorns starfish (Acanthaster planci) populations in the Indian and Pacific Oceans

Spatial variation in allelic frequencies at nine allozyme loci were assayed in 20 populations of the crown‐of‐thorns starfish, Acanthaster planci, collected throughout the Pacific and Indian Oceans. These data were analyzed together with published data, for the same loci, from an additional 19 populations, giving a total sample size of approximately 1800 individuals. There was a marked discontinuity between the Indian and Pacific Ocean populations, but those off Western Australia and from the Southeast Asian region had a strong Pacific affinity. The genetic groups were congruent with the distributions of two color morph groups: gray‐green to red‐brown forms in the Pacific and a blue to pale red form in the Indian Ocean. These patterns of genetic structure are similar to those described for the starfish Linckia laevigata, which has similar life‐history characteristics. Vicariant events may have influenced some populations within the Pacific, but the allozyme data cannot resolve the effects of these events clearly. Patterns of variation within regions were consistent with isolation by distance, but, at larger scales, were obscured by regional vicariance and some outliers, particularly by apparently high levels of gene flow between Japan and the Great Barrier Reef, Australia. Apparent gene flow between population pairs was not closely related to present‐day ocean currents. The results demonstrate a strong influence of allopatric separation on genetic divergence at large geographic scales, but also show evidence of slow rates of change in gene frequencies consistent with the large population sizes of this species. Low levels of divergence between groups demonstrate the genetic structure is recent (Pleistocene) and are likely responses to changes in climate and sea level.

GENETIC STRUCTURE OF GIANT CLAM (TRIDACNA MAXIMA) POPULATIONS IN THE WEST PACIFIC IS NOT CONSISTENT WITH DISPERSAL BY PRESENT-DAY OCEAN CURRENTS

The Pacific marine biota, particularly species with long planktonic larval stages, are thought to disperse widely throughout the Pacific via ocean currents. The little genetic data available to date has supported this view in that little or no significant regional differentiation of populations has been found over large geographical distances. However, recent data from giant clams has demonstrated not only significant regional differentiation of populations, but routes of gene flow that run perpendicular to the main present‐day ocean currents. Extensive surveys of genetic variation at eight polymorphic loci in 19 populations of the giant clam Tridacna maxima, sampled throughout the West and Central Pacific, confirmed that the patterns of variation seen so far in T. gigas were not unique to that species, and may reflect a fundamental genetic structuring of shallow‐water marine taxa. Populations of T. maxima within highly connected reef systems like the Great Barrier Reef were panmictic (average FST < 0.003), but highly significant genetic differences between reef groups on different archipelagos (average FST = 0.084) and between West and Central Pacific regions (average FST = 0.156) were found. Inferred gene flow was high (Nem usually > 5) between the Philippines and the Great Barrier Reef, between the Philippines and Melanesia (the Solomon Islands and Fiji), and between the Philippines and the Central Pacific island groups (Marshall Islands, Kiribati, Tuvalu and Cook Islands). Gene flow was low between these three sets of island chains (Nem < 2). These routes of gene flow are perpendicular to present‐day ocean currents. It is suggested that the spatial patterns of gene frequencies reflect past episodes of dispersal at times of lower sea levels which have not been erased by subsequent dispersal by present‐day circulation. The patterns are consistent with extensive dispersal of marine species in the Pacific, and with traditional views of dispersal from the Indo‐Malay region. However, they demonstrate that dispersal along present‐day ocean surface currents cannot be assumed, that other mechanisms may operate today or that major dispersal events are intermittent (perhaps separated by several thousands of years), and that the nature and timing of dispersal of Pacific marine species is more complex than has been thought.

Penaeid genetics and biotechnology

The application of biotechnology to penaeid prawn aquaculture is increasing in importance. Early doubts that such approaches were unnecessary, given the crude state of development of the prawn aquaculture industry, have been replaced by the recognition that sophisticated molecular approaches will be required to tackle key problems of disease recognition and control, and to achieve higher production through the development of domesticated strains. Progress in penaeid genetic and biotechnological research has been slow because of a lack of knowledge on fundamental aspects of their biology. However, data is beginning to emerge from research projects started in the last decade, and is likely to increase rapidly in the next 10 years. Highly variable markers have been developed that allow the genetic variation in prawn stocks to be assessed, even in highly inbred cultured lines. The extent to which growth rate is under genetic control has now been assessed in some species using rigorous experimental designs to estimate heritabilities. Highly sensitive techniques that allow the isolation and characterisation of very small quantities of peptides are being used to investigate the endocrine control of reproduction, and work has begun on the isolation and characterisation of genes that play an important role in growth and reproduction. Greater attention is being paid to developing cell lines for prawns, and to the development of molecular probes for the identification of pathogens. These tools will provide a basis for a more detailed description of the penaeid prawn genome, for understanding disease resistance, and for developing effective control of reproduction and the development of disease free or disease resistant domesticated lines.

Mitochondrial DNA variation in Indo-Pacific populations of the giant tiger prawn, Penaeus monodon

Surveys of mitochondrial DNA (mtDNA) variation in the giant tiger prawn, Penaeus monodon, using restriction fragment length polymorphisms have provided the first clear evidence that the Indo‐West Pacific region is a site of accumulation of genetic diversity rather than a site of origin of genetic diversity. No haplotyes were found in common between a group of five southeast African populations and a group of five Australian (including Western Australia) and three southeast Asian populations. The dominant haplotype was different in the Australian and southeast Asian population groups. Genetic diversity (π) was greatest in Indonesia (π averaged 0.05), less in the Philippines and Australia (π averaged 0.01), and markedly less in the southeast African and the West Australian populations (π averaged 0.003). The high diversity of the southeast Asian populations resulted from the occurrence in those populations of a set of haplotypes found only in southeast Asia but derived from the southeast African haplotypes. These genetic variants therefore evolved in the Indian Ocean and later migrated into the Indo‐West Pacific region. Low genetic variation in the geographically marginal populations in southeast Africa and Western Australia is considered to be the result of bottlenecks, but mismatch distributions suggest that large population sizes have been maintained in Indonesian populations for long periods.

Gene flow and population history in high dispersal marine invertebrates: mitochondrial DNA analysis of Holothuria nobilis (Echinodermata: Holothuroidea) populations from the Indo-Pacific

The sea cucumber, Holothuria nobilis, has a long‐lived planktotrophic larvae, and previous allozyme surveys have suggested that high dispersal is realized. In contrast, recent ecological studies indicate that dispersal is low. To reconcile these data, and to investigate the evolution of this Indo‐Pacific species, we screened geographical variation in 559 bp of a mitochondrial gene (COI) in 360 samples from the Australasian region and La Réunion. Sequences from La Réunion differed by > 7% from others and may constitute another species. Haplotype diversity in other samples was high (0.942, SD = 0.007), but haplotypes were closely related (mean nucleotide diversity: 0.0075, SD = 0.0041). amova, pairwise FST values and exact tests did not detect significant population structure. Nested clade analysis showed that one of two main clades was over‐represented in west Australia, whereas the other was more common in the northern Great Barrier Reef. Isolation‐by‐distance was identified as the main determinant of population structure at several clade levels. Contiguous range expansion was inferred for evolutionary older clade levels and this may correspond to a late Pleistocene (88 000–193 000 years ago) population expansion inferred from haplotype mismatch distributions. Thus, the population genetic structures detected are likely to be formed prior to the last ice age, with some indications for high dispersal on shorter time scales.

Genomics in marine monitoring: new opportunities for assessing marine health status

This viewpoint paper explores the potential of genomics technology to provide accurate, rapid, and cost efficient observations of the marine environment. The use of such approaches in next generation marine monitoring programs will help achieve the goals of marine legislation implemented world-wide. Genomic methods can yield faster results from monitoring, easier and more reliable taxonomic identification, as well as quicker and better assessment of the environmental status of marine waters. A summary of genomic methods that are ready or show high potential for integration into existing monitoring programs is provided (e.g. qPCR, SNP based methods, DNA barcoding, microarrays, metagenetics, metagenomics, transcriptomics). These approaches are mapped to existing indicators and descriptors and a series of case studies is presented to assess the cost and added value of these molecular techniques in comparison with traditional monitoring systems. Finally, guidelines and recommendations are suggested for how such methods can enter marine monitoring programs in a standardized manner.

A threat to coral reefs multiplied? Four species of crown-of-thorns starfish

In the face of ever-increasing threats to coral reef ecosystems, it is essential to understand the impact of natural predators in order to devise appropriate management strategies. Destructive population explosions of the crown-of-thorns starfish Acanthaster planci have devastated coral reefs throughout the Indo-Pacific for decades. But despite extensive research, the causes of outbreaks are still unclear. An important consideration in this research is that A. planci has been regarded as a single taxonomic entity. Using molecular data from its entire distribution, we find that A. planci is in fact a species complex. This discovery has important consequences for future coral reef research, and might prove critical for successful reef conservation management.

Zoanthids (Anthozoa, Hexacorallia) from the Great Barrier Reef and Torres Strait, Australia : systematics, evolution and a key to species

Abstract. Zoanthid taxonomy is currently in a state of chaos, with many described species very few of which can be reliably identified. As part of a genetically based, objective reappraisal of the number of northern Australian species, a total of 355 zoanthid specimens were collected from 19 localities in the Great Barrier Reef and Torres Strait during 1992–1994. Specimens, initially assigned to one of ten morphological or ecological forms, were subjected to allozyme electrophoretic analysis. Analysis of genetic data revealed only seven discrete (i.e., non-interbreeding) groups in the family Zoanthidae. These groups, which are delimited by fixed gene differences, are considered species under a biological species concept. Some species show considerable morphological variation and have broad environmental tolerances. We provide the first key to Great Barrier Reef zoanthid species based on our results and observations. Species can be differentiated in the field on the basis of gross colony morphology, sand encrusting habit, polyp form and habitat. Genetic data are also used to generate a phylogenetic hypothesis of relationships among the Zoanthidae, which is compared to previous morphologically based systems. Division of the group on the basis of mesenterial arrangement appears justified, but nematocyst data appear less valuable in phylogenetic studies of the group than has been suggested previously.

PmLT, a C-type lectin specific to hepatopancreas is involved in the innate defense of the shrimp Penaeus monodon

A diverse class of proteins called lectins plays a major role in shrimp innate immunity. In this study, the cDNA encoding a C-type lectin of Penaeus monodon (PmLT) was cloned, and its potential role examined. Despite the low overall amino acid sequence identity with other animal lectins, PmLT includes conserved carbohydrate recognition domains (CRDs) characteristic of animal C-type lectins. Unlike the other two P. monodon lectin-like proteins described to date that have one CRD, PmLT has two CRDs. The first CRD contains a QPD motif with specificity for binding galactose, while the second CRD contains a EPN motif for binding mannose. PmLT transcripts can be detected in the hepatopancreas but not in other tissues. Expression studies showed that PmLT mRNA transcript level decreased initially and then gradually increased after whole shrimp or hepatopancreas tissue fragments were treated with white spot syndrome virus (WSSV) extract but were not affected by bacteria. Using anti-rPmLT antibody, PmLT was detected only in the hepatopancreas specific F cells (Hpf). In vitro encapsulation assay showed that agarose beads coated with rPmLT were encapsulated by hemocytes indicating a role in innate immune response. In summary, PmLT is produced in the hepatopancreas and may act as a pattern recognition protein for viral pathogens and also activates the innate immune responses of the shrimp to bacteria. The dual-CRD structure of PmLT may assist the recognition of diverse pathogens.