Assessing the ecological impacts of an introduced seastar: the importance of multiple methods

Introduced species are having major impacts in terrestrial, freshwater, and marine ecosystems worldwide. Given that resources for management are limited and that only a small percentage of invaders are likely to cause large ecological change, management priorities should be based on the severity of immediate and anticipated impacts on native assemblages and commercial species. This paper synthesizes work on the current and predicted impacts of an introduced predatory seastar (Asterias amurensis) on soft sediment assemblages, including native species subject to commercial fishing, in the Derwent Estuary and other areas of southeast Tasmania. Due to the absence of baseline data prior to the arrival of the seastar and the presence of other anthropogenic stressors in the estuary, estimating the impact of the seastar is difficult. To help overcome the weaknesses of any single method, our assessment of impact rests on 'weight of evidence' from multiple approaches. Results from experimental manipulations at small scales, detailed observations of feeding, and field surveys over a range of spatial scales in areas with and without the seastar provide strong evidence that predation by the seastar is likely to be responsible for the decline and subsequent rarity of bivalve species that live just below or on the sediment surface in the Derwent Estuary. The data suggest that should seastar densities in other areas on the Tasmanian coast attain the current levels in the Derwent Estuary, there are likely to be large direct effects on native assemblages, particularly on populations of large surface dwelling bivalves, including several commercial species. Given the seastar's ability to exploit a broad range of food resources other than bivalves, and the functional importance of bivalves in native systems, we predict broader direct and indirect effects on native assemblages. We would be unable to reach these same conclusions from a single approach to assessing impacts. The overall picture from the combination of methods at different scales provides more information than the sum of the results of the separate lines of investigation.     

Differentiation of Paenibacillus larvae subsp. larvae,the cause of American foulbrood of honeybees,by using PCR and restriction fragment analysis of genes encoding 16S rRNA

A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.     

Variability in the impact of an introduced predator (Asterias amurensis: Asteroidea) on soft-sediment assemblages

The introduction and establishment of the predatory asteroid Asterias amurensis in coastal waters of southern Australia is considered a major threat to benthic marine assemblages and commercial bivalve species. We compare the impact of the seastar in experiments on three soft-sediment assemblages. The experiments were carried out immediately beyond the current range of the seastar in southeast Tasmania. This allows us to assess the repeatability, and hence predictability, of the type and magnitude of the impact of A. amurensis on soft-sediment assemblages. Responses to manipulations at the species level were dissimilar across the three experiments, reflecting marked initial differences in the three assemblages at both the species and functional group levels. However, at the functional group level, there were notable similarities in the impact of the seastar on the different assemblages. When potential prey taxa were separated into functional groups reflecting ecological availability (e.g. surface dwelling vs. deep burrowing bivalves), seastar effects were largely restricted to the surface bivalves. While the effect of seastar predation on surface bivalves was common in all three assemblages, the magnitude of the impact varied both among and within assemblages. Variability in the impact of A. amurensis at both levels appeared to be largely due to differences in relative availability of prey species. Overall, the results of these experiments indicate that while the impact of the seastar is broadly predictable at the functional group level, the exact nature of seastar effects is likely to be site- and time-specific given the inherent natural variability in soft-sediment assemblages and the seastar's responses to them.     

Discrimination of two Japanese water pennies,Eubrianax granicollis Lewis and E. ramicornis Kiesenwetter (Coleoptera: Psephenidae), based on laboratory rearing and molecular taxonomy

Two psephenid beetles, Eubrianax granicollis Lewis and E. ramicornis Kiesenwetter, are common species on the main islands of Japan (i.e. Honshu, Shikoku and Kyushu), but diagnostic characters for larval identification are unknown. Two types of field‐collected Eubrianax larvae from Honshu and Kyushu were discriminated based on the distributions of granules on the dorsal surface. These larvae were assigned to E. granicollis and E. ramicornis by comparing them with larvae of the two species obtained via laboratory rearing. The two species were also identified unambiguously on the basis of their mitochondrial cytochrome oxidase subunit I (COI) gene sequences. Larval and pupal morphology are described based on laboratory‐reared specimens.     

DNA barcoding of eight North American coregonine species

Coregonine fishes have a circumpolar distribution in the Arctic and sub-Arctic Northern Hemisphere. This subfamily of Salmonidae consists of three genera: Prosopium, Stenodus and Coregonus, including over 30 species. Many species overlap spatially and are difficult to distinguish based on morphological characteristics, especially as larvae or juveniles. Here we present a method for rapid and cost-effective species identification for representatives of the three genera based on sequence variation at the mitochondrial cytochrome c oxidase subunit I gene (COI). We examined eight species common to North America with distributional overlap in Alaska. Mean pairwise sequence divergence for all eight species was 7.04% and ranged from 0.46% to 14.23%. This sequence variation was used to develop a genetic assay based on restriction fragment length polymorphism. In a blind test, this assay provided correct species assignment for 48 of 49 individuals representing all eight species. The single incorrect assignment may reflect hybridization between two closely related species. This DNA barcode-based assay promises to aid fishery managers and researchers by providing a cost-effective alternative to large-scale sequence analysis for identification of North American coregonine fishes.     

Identification of weakfish Cynoscion (Gill) in the Bay of Panama with RFLP markers

A molecular approach, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), was developed to identify the planktonic larvae of Cynoscion species. Species-specific mitochondrial DNA markers were developed using three restriction endonucleases ( Dde I, Hae III and Hin fI). These markers permitted the accurate discrimination of the five Cynoscion species in the Bay of Panama.     

Molecular Diet Analysis of Phyllosoma Larvae of the Japanese Spiny Lobster <Emphasis Type="Italic">Panulirus japonicus</Emphasis> (Decapoda: Crustacea)

To clarify the natural diet of phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus, the sources of 18S rDNA clones obtained from the hepatopancreas were investigated. Of a total of 1537 clones examined, 160 had different restriction profiles from the host larvae, in which 21 restriction types were observed. Nucleotide sequences of 16 of 21 restriction types were successfully determined and their assignments were investigated by homology search and phylogenetic analysis. From seven late-stage larvae collected in spring to early summer, eukaryote DNA molecules of Teleostei, Oomycetes, Mycetozoa, and Fungi were identified. Exogenous DNA from four younger phyllosoma larvae collected in late autumn could not be recovered. A previous study identified DNAs of cnidarians and urochordates in late-stage phyllosoma larvae of a closely related species collected in winter. This indicates that the phyllosoma larvae are opportunistic carnivores, whose diets correlate with the relative abundance of prey organisms in the ambient water.     

High‐throughput species identification: from DNA isolation to bioinformatics

Ichthyoplankton collections provide a valuable means to study fish life histories. However, these collections are greatly underutilized, as larval fishes are frequently not identified to species due to their small size and limited morphological development. Currently, there is an effort underway to make species identification more readily available across a broad range of taxa through the sequencing of a standard gene. This effort requires the development of new methodologies to both rapidly produce and analyse large numbers of sequences. The methodology presented in this paper addresses these issues with a focus on the larvae of large pelagic fish species. All steps of the methodology are targeted towards high‐throughput identification using small amounts of tissue. To accomplish this, DNA isolation was automated on a liquid‐handling robot using magnetic bead technologies. Polymerase chain reaction and a unidirectional sequencing reaction followed standard protocols with all template cleanup and transferring also automated. Manual pipetting was thus reduced to a minimum. A character‐based bioinformatics program was developed to handle the large sequence output. This program incorporates base‐call quality scores in two types of sample to voucher sequence comparisons and provides suggested identifications and sequence information in an easily interpreted spreadsheet format. This technique when applied to tuna and billfish larvae collected in the Straits of Florida had an 89% success rate. A single species (Thunnus atlanticus) was found to dominate the catch of tuna larvae, while billfish larvae were more evenly divided between two species (Makaira nigricans and Istiophorus platypterus).     

Molecular Species Identification of Newly Hatched Hawaiian Amphidromous Gobioid Larvae

This report describes a method for the determination of species identity of newly hatched larvae of five sympatric Hawaiian amphidromous gobioids (Lentipes concolor, Sicyopterus stimpsoni, Awaous guamensis, Stenogobius hawaiiensis, and Eleotris sandwichensis). Polymerase chain reaction (PCR) was used to amplify a homologous section of the cytochrome b (Cyt b) region of the mitochondrial genome (mtDNA) from adults of all five species. The resulting PCR-amplified DNA was subjected to restriction fragment length polymorphism (RFLP) analysis producing species-specific restriction patterns. PCR products from the five species were sequenced to substantiate correct amplification, restriction site locations, and fragment sizes. The sequence data were also used to construct a phylogenetic tree. Individual, newly hatched, wild-caught larvae of amphidromous gobioids of unknown species affinity were sorted into six morphotypes based on physical characteristics. These typed larvae and those from two species that spawned in captivity were subjected to the same molecular analysis as the adults. The RFLP results from adults and larvae were compared, allowing larval morphotypes to be assigned to the appropriate species. These comparisons permitted construction of an identification key to the newly hatched larvae of these species based solely on physical characteristics for use in future field studies. Received April 29, 1998; accepted September 30, 1998.     

猕猴桃属植物叶绿体基因PCR-RFLP分析

分别用2个不同限制性内切酶对猕猴桃属27个种和15个栽培品种的叶绿体基因(rbcL基因和psbA基因)的PCR扩增产物进行酶切分析,共得到25个限制性位点,其中24个具有多态性。确立了该属植物的单元型分布,对该属植物的系统发育方式和部分重要种类的亲缘关系进行了探讨,拓展了可用于该属植物分子系统学研究的遗传信息。     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.     

Diagnostic DNA amplification from individual tick eggs,larvae and nymphs

The species of single tick eggs, larvae and nymphs was determined by PCR amplification and characterization of the hypervariable, second transcribed spacer (ITS2) of the multicopy ribosomal RNA gene (rDNA). Engorgement of larvae and nymphs did not preclude species identification. The method is generally applicable for ixodid and argasid ticks and can be used for epidemiological studies requiring the identification of individuals from pre-adult stages.     

A technique for identifying the roots of different species in mixed samples using nuclear ribosomal DNA

Abstract. Question: Is it possible to determine the species composition of root samples containing multiple species, without first disentangling individual roots? Methods: The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified and sequenced from four California annual grassland species (two Poaceae and two Asteraceae). Restriction enzymes that cut the ITS region of each species into uniquely sized fragments were identified based on DNA sequence variation of the ITS regions. Mixed root samples were analysed to test the ability of the method to identify the presence or absence of each species in multi‐species samples. Results: The technique successfully identified species present in multi‐species samples. ITS regions were shorter in Poaceae than in Asteraceae, so size differences alone were sufficient to distinguish these taxonomic groups. At the species level, digestion of ITS regions with the appropriate restriction enzymes yielded at least one uniquely sized fragment for each species. Conclusions: This method is the first to identify the species composition of mixed root samples. It should be applicable to most plant species because the ITS region is flanked by universal primers and most species have unique ITS sequences. The ability to determine species‐specific rooting distributions has broad applications in vegetation science.     

Characterization of Adoxophyes honmai single-nucleocapsid nucleopolyhedrovirus: morphology,structure, and effects on larvae

A nucleopolyhedrovirus (NPV) was isolated from a diseased larva of the smaller tea tortrix, Adoxophyes honmai, collected from a tea field in Tsukuba, Ibaraki, Japan. Electron microscopic observations confirmed that A. honmai NPV (AdhoNPV) was a single-nucleocapsid type virus. The genome size of AdhoNPV was estimated to be 111.6 +/- 0.9kb (mean +/- SE) by restriction endonuclease analysis. AdhoNPV was also infectious to two other Adoxophyes species, the summer fruit tortrix Adoxophyes orana and Adoxophyes dubia. The LD50 values for neonatal, second, third, fourth, and fifth (final) instar larvae of A. honmai were determined as 61, 107, 688, 1,961, and 4,085 occlusion bodies/insect, respectively. Most of the infected larvae died 5-9 days after molting to the final instar, regardless of the timing of inoculation. However, when neonates were exposed to extremely high doses of AdhoNPV (greater than 100 x LD90), larval development was prevented and most of the larvae died in the first instar.     

Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

Pathogenicity of diatraea saccharalis Densovirus to Host Insets and Characterization of its Viral Genome

Pathogenicity of the Diatraea saccharalis densovirus (DsDNV) was tested on its host larvae. The results showed that up to 4 days after inoculation, no larvae mortality was observed and the infected larvae started to exhibit the infection symptoms from the fourth day. After 5 days of infection, the cumulative mortality of infected larvae increased significantly and reached 60% after 12 days and 100% after 21 days of infection, whereas that of the control group was only 10% and 20%, respectively, after same periods of infection, suggesting that the high mortality of infected larvae groups was due to the high pathogenicity of DsDNV. The size of the DsDNA was determined by Electron microscopy visualization of viral DNA molecules and gel electrophoresis of both native and endonuclease digested DNA fragments. The total length of the native DsDNA was about 5.95 kb. The DsDNV DNA was digested with 16 restriction enzymes and a restriction map of those enzymes was constructed with 41 restriction sites. Comparison of the restriction map of the DsDNV genome with those of the genomes ofJunonia coenia densovirus (JcDNV) and Galleria mellonella densovirus (GmDNV) indicated that the three densovirus genomes were found to share many identical restriction sites. Thus, most of the restriction sites of the following endonucleases Bam H I, Hha I, Xba I, Cla I, Asp 700, Spe I, Nco I and Bcl I, were found to be conserved among the three densovirus genomes. Symmetrical cleavage sites mapped at the both ends of the genome suggested the presence of inverted terminal repeats (ITRs) whose size was estimated to be about 500 bp. The similar genome size, almost identical restriction sites and presence of an ITR of about 500 bp for these three densoviruses suggested that they belong to the same group of ambisense densoviruses.     

Molecular identification of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) from fish and cetacean in Japanese waters

Parasites morphologically consistent with Anisakis simplex sensu lato collected from the coast of Japan and Western North Pacific Ocean were examined by PCR-RFLP of the ITS region (ITS1, 5.8 subunit rRNA gene and ITS2) and mtDNA cox1. The RFLP patterns of rDNA generated by HinfI and HhaI showed that 100% of the larvae collected from Hokkaido and 94% of adults collected from Western North Pacific Ocean were identified as A. simplex sensu stricto. On the other hand, 97% of the larvae collected from Fukuoka prefecture were identified as A. pegreffii. A hybrid genotype was found in adults in Western North Pacific Ocean and larva in Fukuoka prefecture. These findings revealed that A. simplexs. str. is primarily distributed in the North Pacific Ocean and A. pegreffii is primarily distributed in the southern Sea of Japan. RFLP analysis of mtDNA cox1 showed different patterns between A. simplex s. str. and A. pegreffii after digestion with HinfI. This polymorphism obtained by RFLP analysis of mtDNA cox1 proved the usefulness as new genetic markers to distinguish two sibling species.     

DNA barcoding reveals the temporal community composition of drifting fish eggs in the lower Hongshui River,China

Determining the temporal community composition of fish eggs in particular regions and understanding the reproductive times of regional fish taxa are key aspects of the management and regulation of regional fish stocks. However, it is extremely difficult to accurately identify fish eggs due to the absence of diagnostic morphological characters. We sampled fish eggs in the lower Hongshuihe River (an upper mainstem of the Pearl River) between May and September 2020. We then used DNA barcoding to determine the species composition of the egg pool and to predict the spawning periods of the identified species. A total of 641 eggs and 17 larvae were chosen for molecular identification; 397 eggs and 17 larvae yielded high‐quality barcoding sequences. The high failure rate (~38%) was most likely due to long‐term storage in low concentrations of ethanol prior to molecular analysis. We successfully classified 392 eggs into 10 species and 13 larvae into four species using public databases. Most of the species identified in the egg pool were small and/or benthic, and migratory species were rare. This may partially reflect the adverse effects of hydropower cascade development in this river section. We also found that spawning periods tended to be species‐specific. Our study provides a reference for the conservation and management of regional fishery stocks.     

Isolation of organellar DNA from Codium fragile (Codiaceae, Codiales, Ulvophyceae)

Organellar DNA was isolated from Codium fragile (Suringar) Hariot (Codiaceae, Codiales, Ulvophyceae) by CsCI-buoyant density centrifugation in the presence of Hoechst dye 33258. Three bands were formed by ultracentrifugation and each fraction of DNA was identified by Southern hybridization. The uppermost fraction was identified as chloroplast DNA, the middle fraction was nuclear DNA and the bottom fraction was mitochondrial DNA. Nuclear rDNA was isolated in the same fraction as mitochondrial DNA. The estimated genome size of mitochondrial DNA by analysis with restriction endonucleases was more than 141.6 kb, which was larger than that of microalgae but smaller than land plants. Restriction endonuclease analysis of the chloroplast DNA showed no difference with that known of C. fragile in New York.