Single-step nested multiplex PCR to differentiate between various bivalve larvae

A nested multiplex polymerase chain reaction (PCR) assay has been developed that allows the discrimination between six bivalve larvae common to Danish coastal waters (Cerastoderma edule, Macoma balthica, Mytilus edulis, Spisula subtruncata, Ensis americanus and members of the order Myoida). This assay involves the simultaneous use of a pair of general universally targeted 18S rRNA gene primers, five specific 18S rRNA gene targeted oligonucleotide primers internal (nested) to the universal primer pair and one species-specific primer that is not nested (Mya). The specificity of each primer was evaluated in silico, empirically, and verified further by sequencing of amplification products from single larvae collected from plankton samples. Identification of individually isolated bivalve larvae from plankton samples was based on the size of the PCR product produced by the specific primers after visualisation by agarose gel electrophoresis. Preliminary studies indicated that this method was suitable for use with freshly collected and preserved larvae, and is therefore suitable for field application.Communicated by M. Kühl, Helsingør     

Differential ingestion of zooplankton by four species of bivalves (Mollusca) in the Mali Ston Bay,Croatia

This study provides information about differences in composition of ingested zooplankton amongst bivalve species coexisting in the same area in a period from May 2009 to December 2010. The study was conducted at the Mali Ston Bay (42°51′ N, 17°40′ E)—the most important bivalve aquaculture area in the eastern Adriatic Sea. Stomach content analysis was performed on cultured species—Ostrea edulis and Mytilus galloprovincialis, and commercially important bivalve species from their natural environment—Modiolus barbatus and Arca noae. Results confirmed carnivory in bivalves, both from natural and cultured populations, but cultured species had higher numbers of zooplankters than those living on the seabed. The most abundant taxa were bivalve larvae, followed by tintinnids, copepods, unidentified eggs and gastropod larvae. Recorded numbers of bivalve larvae in M. galloprovincialis stomach were the highest so far reported and show that mussels impact the availability of natural spat.     

Comparative population genetic structure of marine gastropods (Littorina spp.) with and without pelagic larval dispersal

 Population genetic theory predicts that marine animal species with planktonic larvae will have less genetic structure than those with direct development. We compared the genetic structure of four species of littorinid snails – two with planktonic egg capsules that hatch as planktonic larvae and two with benthic egg masses that hatch as crawl-away juveniles. We used DNA sequencing and single stranded conformational polymorphism (SSCP) to assess sequence variation in a 480 bp fragment of the mitochondrial cytochrome b gene and then used an analysis of molecular variance (AMOVA) to estimate Φ_st for populations from the northeastern Pacific coast. One of the two direct-developing species, Littorina subrotundata, had a moderate amount of population structure (Φ_st=0.209) as expected but the other direct-developing species, L. sitkana, was nearly fixed for a single haplotype that made it impossible to precisely estimate Φ_st. One of the two planktonic-developing species, L. scutulata, did not show any significant population structure (Φ_st=0.004). In contrast to our expectations, the other planktonic-developing species, L. plena, showed some weak but statistically significant population structure (Φ_st=0.052). We discuss how differences in population genetic structure between species with the same type of development may reflect differences in their historical demography. Received: 22 December 1999 / Accepted: 24 July 2000     

<Emphasis Type="Italic">Cochlodinium polykrikoides</Emphasis> blooms and clonal isolates from the northwest Atlantic coast cause rapid mortality in larvae of multiple bivalve species

Globally, many commercial bivalve populations have declined in recent decades. In addition to overharvesting and habitat loss, the increasing frequency and intensity of harmful algal blooms (HABs) are likely to contribute to bivalve losses, particularly in cases where blooms negatively impact larval stages. This paper reports on the lethal effects of clonal cultures and blooms of Cochlodinium polykrikoides from the US Atlantic coast on the larvae of three species of commercially and ecologically valuable bivalves: the Eastern oyster (Crassostrea virginica), the bay scallop (Argopecten irradians), and the Northern quahog (hard clam; Mercenaria mercenaria). Both cultures and blooms of C. polykrikoides were highly toxic to all three species of bivalve larvae causing 80–100% mortality during 24- to 72-h exposures at concentrations of 1–2 × 10³ cells ml⁻¹. Toxicity was dependent on cell densities, growth stage of C. polykrikoides (i.e. cultures in exponential stage growth were more toxic than later stages), exposure time of larvae to cells (i.e. longer exposure caused higher mortality), the age of larvae (i.e. younger larvae were more sensitive), and the relative abundance of C. polykrikoides (i.e. the presence of other microalgae decreased toxicity). Free radical-scavenging enzymes (peroxidase and catalase) and the removal of C. polykrikoides cells (i.e. culture filtrate) significantly increased larval survival suggesting toxicity is maximized by contact with live cells and may involve labile toxins bound by these compounds including e.g. reactive oxygen species. The toxicity of C. polykrikoides to bivalve larvae was generally more severe than other HAB species (e.g. Karenia brevis, Karlodinium veneficum, Alexandrium tamarense, Prorocentrum minimum). Since the bivalves in this study spawn in the months when C. polykrikoides blooms on the east coast of North America, these results suggest that these blooms may have detrimental effects on efforts to restore these already diminished populations.     

Effect of the receding ice-edge on the condition of mid-water fishes in the northwestern Weddell Sea: results from biochemical assays with notes on diet

Electrona antarctica and Bathylagus antarcticus are dominant fishes in the Southern Ocean pelagic ecosystem with disparate life histories, making them excellent subjects for comparative studies. Twenty-one indices of nutritional condition were compared in both species by observing changes in each index as it varied spatially and temporally in association with the marginal ice-zone bloom in the northwestern Weddell Sea. E. antarctica and B. antarcticus exhibited very different patterns of response to the bloom. Nutritional condition increased in 11 of 21 measures in E. antarctica, suggesting that it was in poorer condition at ice-covered stations and in greatly improved condition in post-bloom, open-water stations. The data, combined with a few observations of larvae, indicate that E. antarctica increases feeding before the spring bloom, rapidly building stores of lipid, which are probably used for spawning. In contrast, only three measures of condition were variable in B. antarcticus, and lipid actually decreased from ice-covered to open water. RNA:DNA values increased in open water, coinciding with an increase in food volume present in their guts. These observations, coupled with observations of larvae in ice-covered stations, suggest that B. antarcticus possesses sufficient energetic stores to spawn prior to the bloom, so that larvae are able to fully utilize available resources associated with the spring bloom. Increased somatic growth, as indicated by RNA:DNA, may not begin until later, when the bloom was beginning to decline. Earlier work, which suggested that the deeper living, non-migrating species, like B. antarcticus, would not be affected by the bloom until after shallower, migratory species, like E. antarctica, is supported. Of the assays tested, RNA:DNA appeared to be the most sensitive. The combined assessment of many measures including biochemical, compositional, dietary, and age estimates from single specimens is possible if the samples are treated with sufficient care. The reliance upon a single measure to help interpret the ecology of a species, especially in nekton-sized species, is not as effective as techniques used in combination. Received: 12 January 2000 / Accepted: 16 August 2000     

A functional interpretation of cilia and mucocyte distributions on the abfrontal surface of bivalve gills

The lack of fundamental data on the abfrontal surface of bivalve gills has prompted a comparative study of cilia and mucocytes on this surface, using scanning electron microscopy and histology on eight species of bivalves, representing seven families and the four major gill types: Mytilus edulis, Modiolus modiolus, Arca zebra, Placopecten magellanicus, Crassostrea virginica, Spisula solidissima, Mercenaria mercenaria, and Mya arenaria. Abfrontal cilia and mucocytes were found in all species studied, with types and densities differing within and between gill types. The three species of homorhabdic filibranchs presented different densities of abfrontal cilia and mucocytes, from very dense in M. edulis to sparse in A. zebra. The heterorhabdic gills had intermediate cilia and mucocyte densities, with highest concentrations of both abfrontal cilia and mucocytes on the principal filaments. The eulamellibranchs showed low ciliary densities together with high mucocyte densities, especially in S. solidissima, where the abfrontal mucocytes were glandular. These results indicate that: (1) the abfrontal surface is a vestigial mucociliary surface; (2) the abfrontal surface cannot participate in water pumping in most species, due to low ciliary densities; and (3) species with high densities of abfrontal mucocytes could utilize abfrontal mucus to reduce drag, especially in the highly fused gills, such as those of the eulamellibranchs. The differing distributions of abfrontal cilia and mucocytes may reflect different selective pressures acting on the gills within the various taxa. Received: 12 February 2000 / Accepted: 10 September 2000     

Comparative morphology and cytology of siphons and siphonal sensory organs in selected bivalve molluscs

 The form, anatomy and cytology of the water-passages in siphon-possessing and siphon-lacking species of a selected group of bivalve molluscs from the Red Sea (Callista florida, Circe currogata, C. crocea, Lucinia dentifera, Dosinia histrio, Pitar hebraea, Tridacna maxima, Pteria aegyptica), and from the Mediterranean Sea (Spondylus spinosus, Pinctada radiata, Pinna nobilis, Donax trunculus, D. semistriatus, Mactra stultorum, Tapes decussatus, Petricola lithophaga, Brachidontes pharaonis) were studied by means of light and electron microscopy. In the mytiloids, ostreoids and ptrioids studied, the water-passages are covered by a ciliated epithelium and the few tentacles around their openings are identical to those found on the mantle edges. Contrary to this, in the veneroids studied, the siphons are covered by a microvillar epithelium and their tentacles, especially of the inhalant siphon, reveal characteristic branching. The so-called Type I, Type II and Type III ciliated sensory organs on and within the siphons are similar to those observed in other bivalves, differing from each other in the dimension, number and form of cilia. In the studied mytiloid species only Type I and Type II organs were found. The form and structure of the siphons and the distribution of sensors on them can be used as markers for ecological as well as phylogenetic considerations. Received: 28 January 2000 / Accepted: 28 May 2000     

Molecular studies on the ouabain binding site of the Na+, K+-ATPase in milkweed butterflies

Summary. The Na⁺, K⁺-ATPase of the Monarch butterfly (Danaus plexippus) is insensitive to the inhibition by cardiac glycosides due to an amino acid replacement: histidine instead of asparagine at position 122 of the α-subunit representing the ouabain binding site. By PCR amplification of the DNA sequence of this site, a PCR product of 270 bp was obtained from DNA extracted from Danainae species (Danaus plexippus, D. chrysippus, D. gillipus, D. philene, D. genutia, Tirumala hamata, Euploea spp., Parantica weiskei, P. melusine), Sphingidae (Daphnis nerii) and mimics of milkweed butterflies (Hypolimnas missipus, Limenitis archippus and L. arthemis, Nymphalidae). Analysis of the nucleotide sequences revealed that the single point mutation in the ouabain binding domain (AAC-Asn for CAC-His) was present only in Danaus plexippus, but not in the other species investigated. Since these milkweed butterflies also store cardenolides, other structural modifications of the Na⁺, K⁺-ATPase may have occurred or other strategies of cardenolide tolerance have been developed. Received 15 May 2000; accepted 29 June 2000     

Inhibition and facilitation of bryozoan and ascidian settlement by natural multi-species biofilms: effects of film age and the roles of active and passive larval attachment

Sessile marine invertebrate larvae can recognize suitable settlement substrata by using various environmental cues, including organic/microbial biofilms. In laboratory choice assays, the effect of biofilms of varying ages on the settlement behaviour of two fouling organisms was assessed. The species included the arborescent cheilostome bryozoan Bugula flabellata (Thompson) and the solitary ascidian Ciona intestinalis (L.), both of which are characteristic of temperate sublittoral hard substratum assemblages in northwest Europe. Experiments were carried out using polystyrene petri dish substrata preconditioned with multispecies biofilms from natural laboratory-aquarium seawater for 1, 3, 6 or 12 d. Unfilmed (new, initially sterile) dishes were used as control substrata. Whereas the coronate larvae of B. flabellata generally were inhibited by biofilming, irrespective of film age, the settlement of tadpole larvae of C. intestinalis was facilitated on biofilmed substrata, and numbers of settled larvae generally increased with biofilm age: the highest mean numbers were counted on 12 d-old biofilms. In C. intestinalis, settlement and metamorphosis are processes which can be temporally separate and are possibly induced by different environmental cues. This study therefore distinguished between C. intestinalis larvae which were attached to the biofilm surface by the anterior, and those larvae entrapped by the biofilm but not settled in the conventional meaning of the term. As reported in previous studies, we did observe that such entrapped larvae could subsequently attach and develop successfully into sessile juveniles. Both the numbers of “attached” and “trapped” tadpoles increased with biofilm age. Assuming that “settlement” is essentially a process involving the active behavioural response of larvae to environmental cues, it seems that the facilitated attachment of C. intestinalis onto biofilmed substrata is due to the combined effect of active habitat selection and passive deposition/“entrapment” of larvae onto the “sticky” substratum. Received: 21 August 1996 / Accepted: 21 November 1996     

Behaviour of echinoid larvae around sharp haloclines: effects of the salinity gradient and dietary conditioning

We examined larval response to a range of sharp haloclines and determined the effect of dietary conditioning on that response in the sea urchins Echinometra lucunter and Arbacia punctulata. We reared larvae in the laboratory under a high or low concentration of either single (Isochrysis galbana) or mixed (Isochrysis galbana, Dunaliella tertiolecta, Thalassiosira weissflogii) microalgal species. For both species of sea urchins, rate of larval development was faster and age-specific larval length and width were greater in high-ration than low-ration diets. We examined the distribution of two- and four-arm larvae of E. lucunter from each diet treatment and of four-arm larvae of A. punctulata from the high-ration diets in cylinders with experimentally constructed haloclines. In three of the halocline treatments, the salinity of the bottom layer was 33‰ and that of the top layer was 21, 24 or 27‰ (21/33, 24/33 and 27/33) and in a fourth one, the salinities of the bottom and top layer were 30 and 21‰, respectively (21/30). The position of larvae in the cylinders varied with the steepness of the halocline and with dietary conditioning for both sea urchin species and all developmental stages tested. Significantly more larvae crossed the haloclines into water of 24 and 27‰ salinity than into water of 21‰ salinity. We observed an effect of diet on the position of larvae in the cylinders, and that effect varied among halocline treatments for both species. The proportion of larvae of E.lucunter that crossed the halocline was greater in low- than high-ration diets in the 24/33 and 27/33 treatments. Position of four-arm larvae in the cylinders also varied with food quality in high-ration diets: for E.lucunter in the 24/33 treatments, and for A. punctulata in the 21/30 treatments, more larvae from the single- than from the mixed-species diets were present above the halocline. Salinity in the adult habitat during most of the active reproductive period ranged from 15 to 40‰. We showed that larvae can respond to gradients in salinity, and therefore can remain within a water mass of higher salinity overlying the adult habitat. However, survival of poorly fed larvae may be increased if they are introduced into a new water mass and carried away from a nutritionally poor environment. Received: 9 July 1997 / Accepted: 12 January 1998     

Settlement-competency periods of larvae of three species of scleractinian corals

The pattern of settlement over time of three broadcast spawning coral species (Cyphastrea serailia, Acanthastrea lordhowensis, and Goniastrea australensis) from the Solitary Islands (30°00′S; 153°20′E) was studied in 1995 and 1996 in order to determine the maximum length of time these larvae could remain in the water column and still retain the ability to settle and metamorphose. Larvae were maintained in aquaria and the number which had settled on biologically-conditioned tile pairs was monitored every 5 to 10 d. While the majority of larvae settled quickly after becoming competent, some larvae survived and settled for extended periods after spawning. Competency periods ranged from 26 d for C. serailia to 56 d for G. australensis and 78 d for A. lordhowensis. These data greatly extend the known competency periods for larvae of broadcast-spawning corals and indicate the potential for transport of broadcast-spawned coral larvae over large distances. Medium to long-distance larval dispersal of the species studied provides a mechanism for their widespread distribution in subtropical regions, on reefs which are often widely spaced and relatively isolated. Received: 27 May 1997 / Accepted: 27 November 1997     

Simple molecular method to distinguish the identity of Calanus species (Copepoda: Calanoida) at any developmental stage

Diagnostic morphological characteristics of copepods of the genus Calanus are restricted largely to minor variations in secondary sex characteristics. This presents a persistent problem in the identification of individuals to species level, especially for immature stages. We have developed a simple molecular technique to distinguish between the North Atlantic Calanus species (C. helgolandicus, C. finmarchicus, C. glacialis and C. hyperboreus) at any life stage. Using the polymerase chain-reaction (PCR), the mitochodrial large subunit (16S) ribosomal RNA (rRNA) gene was amplified from individual copepods preserved in ethanol. Subsequent digestion of the amplified products with the restriction enzymes DdeI and VspI, followed by electrophoretic separation in 2% agarose (Metaphor, FMC Ltd), produced a characteristic pattern for each species. The versatility of the method is demonstrated by the unambiguous identification to species of any life stage, from egg to adult, and of individual body parts. Received: 11 May 1998 / Accepted: 5 August 1998     

Population and reproductive biology of two sibling amphipod species from ascidians and sponges

 The population dynamics and reproductive biology of an ascidian- and a sponge-dwelling amphipod were examined. The two undescribed amphipod species, Leucothoe“ascidicola” and L.“spongicola”, are closely related to each other, and occur in ascidians and sponges, respectively, along the Florida Atlantic coast. L. “ascidicola” was abundant in solitary ascidians during fall 1997, disappeared during spring/early summer, and became abundant again in September 1998. During the time when L. “ascidicola” were absent from their hosts, a copepod became a frequent inhabitant of the ascidians but disappeared again when L.“ascidicola” returned to the ascidians in September 1998. The numbers of L.“spongicola” in sponges increased substantially during spring, when high reproductive activity was observed. Following this reproductive peak, both adult and juvenile amphipods apparently left the sponges, and during the summer amphipod numbers in the sponges were very low. Another small amphipod species, which often co-occurred with L.“spongicola”, showed less seasonal variation and was found in sponges throughout the whole study period. The percentage of ovigerous females per host unit was usually lower in the ascidian-dwelling than in the sponge-dwelling amphipods. In solitary ascidians, L.“ascidicola” amphipods usually occurred in groups of several adults, yet there never was more than one ovigerous female per ascidian. In contrast, several ovigerous L.“spongicola” females were found to cohabit in the same spongocoel. This suggests that intrasexual aggression may be stronger among reproductive amphipod females in the ascidians than in sponges. The size distributions of juvenile cohorts indicate that juvenile L.“ascidicola” remain for relatively long time periods in the parental ascidian, where they may reach sexual maturity. In contrast, in L.“spongicola”, only cohorts of very small juveniles could be identified, indicating that juveniles disperse shortly after emerging from the female's brood pouch. It is concluded that extended parental care is of very short duration or does not occur in the sponge-dwelling amphipod L. “spongicola”, possibly because fast-growing sponges with a highly branched spongocoel system do not allow long-lasting coexistence of parent-offspring groups. In contrast, the discrete character of the solitary ascidians may enhance the potential for exclusion of other species, resource monopolization by reproductive females, and furthermore for long-lasting extended parental care in the ascidian-dwelling amphipod. Groups of single parents together with cohorts of large juveniles are reported in the literature for amphipods and isopods from brachiopods, bivalves and ascidians, suggesting that these discrete biotic microhabitats may favor the evolution of extended parental care in peracarid crustaceans. Received: 30 July 1999 / Accepted: 8 May 2000     

Assessment of patch quality by ladybirds: relative response to conspecific and heterospecific larval tracks a consequence of habitat similarity?

Summary. Aphid colonies can reach high levels of abundance but last for short periods of time. The larvae of aphidophagous ladybirds (Coleoptera: Coccinellidae) that feed on these colonies might therefore suffer from starvation, which favours the occurrence of cannibalism and intraguild predation. Thus, the assessment of patch quality becomes crucial and it has been shown that female ladybirds refrain from laying eggs in the presence of an oviposition deterring semiochemical deposited by their larvae. Adalia bipunctata (L.), Adalia decempunctata (L.) and Coccinella septempunctata L. are 3 sympatric species of ladybirds, which can co-occur in aphid colonies. As a consequence, their eggs and larvae are under threat, not only from cannibalism but also intraguild predation. Females should, therefore, also use the tracks deposited by heterospecific larvae to assess the quality of aphid colonies as oviposition sites. The expectation is that: 1- the strength of the reaction to each other’s larval tracks should be correlated with percentage habitat overlap and that 2- the reaction to conspecific larval tracks should be stronger than to heterospecific tracks. In order to test these hypotheses, females’ oviposition behaviour was analysed and a chemical analysis of the tracks of their larvae undertaken. The results show that oviposition behaviour is not related to habitat overlap. Both species of Adalia react to tracks of their own larvae and those of C. septempunctata, but A. decempunctata reacted more strongly than A. bipunctata. C. septempunctata reacted very slightly to its own tracks but not to those of either species of Adalia. The larval tracks are mainly composed of alkanes. Those of the two species of Adalia are qualitatively 100% similar and 60% so when the quantitative results are compared. They are, however, only 24–29% similar to those of Coccinella septempunctata.     

RAPD-PCR investigations of systematic relationships among four species of eels (Teleostei: Anguillidae), particularly Anguilla anguilla and A. rostrata

 We examined the genetic identities of 49 individuals of four species of eels in the genus Anguilla Shaw, A. anguilla, A. japonica, A. reinhardti and A. rostrata, using the random(ly) amplified polymorphic DNA (RAPD)-PCR technique. We used 15 random decamer primers. Out of the 22 148 amplification products detected, 454 markers were evaluated using Nei's distance coefficient, two numerical methods (neighbor-joining, UPGMA), and phylogenetic analysis using parsimony (PAUP). Both numerical and parsimony methods were congruent and confirmed the specific status of all four examined Anguilla species. In all dendrograms, all individuals of each of the species clustered together within four closed groups with highly significant bootstrap values (between 97 and 100%). Additionally 1 to 18 species-specific monomorphic diagnostic fragments were detected in three of the species; none were detected in A. anguilla. Mean interspecific genetic distances ranged from 0.384 to 0.559, with the minimum between A. anguilla and A. rostrata and the maximum between A. anguilla and A. reinhardti. Individuals collected at one locality and belonging to a single species did not cluster together, and neither were any diagnostic monomorphic fragments found for individuals of single localities. This probably reflects a random dispersal of larvae in ocean currents before they reach coastal waters. Among all possible species pairs, only the two Atlantic species were consistently clustered in all dendrograms, with highly significant bootstrap values (100%). Additionally, we detected ten diagnostic markers for this pair of eel species. From RAPD data, we suggest a phylogram which was routed by the descending analysis method: A. reinhardti appears to have been the first species to diverge from a putative common ancestor of the four eel species. Later, A. japonica separated from the hypothetical ancestor of the Atlantic species, which then split recently into A. anguilla and A. rostrata. The close relationship of A. anguilla and A. rostrata is indicated by their low genetic distance (GD = 0.384) and a high degree of shared RAPD fragments (SF = 71.2%); this compares with the following means for all other species pairs: ( = 0.531,  = 44.2%). The prerequisites for using RAPD data for the reconstruction of phylogenies are discussed. Received: 2 September 1999 / Accepted: 8 May 2000     

Real-time PCR assay for detection and relative quantification of <Emphasis Type="Italic">Liocarcinus </Emphasis><Emphasis Type="Italic">depurator</Emphasis> larvae from plankton samples

Accurate species identification of decapod crustacean larvae is required to understand their population distributions, life cycle dynamics and interactions with their habitats. Analysis of plankton samples using morphological taxonomic methods and microscopy is time-consuming, requires highly skilled and trained operatives and may often be inaccurate. As complementary tools to classical identification methods, recent work has focused on the development of molecular approaches and shows their feasibility for species-specific identification. This study has developed real-time PCR assays utilising species-specific Taqman^® probes designed in the cytochrome oxidase I (COI) gene of Liocarcinus depurator, Necora puber, Carcinus maenas and Cancer pagurus. Our study then employed the probe and primers designed for L. depurator to obtain accurate identification and relative abundance estimates of L. depurator larvae in plankton samples collected between March 2005 and October 2006. Ranges of larval abundances were derived from a standard curve created from plankton samples spiked with a known number of larvae reared in the laboratory. Inhibition of the PCR reaction was shown to be an important factor and our results suggested that 0.1 ng of DNA as template provided accurate identification and avoided inhibition. Real-time PCR was shown to provide accurate species identification on unsorted plankton samples and could be suitable for the estimation of larval abundances in the plankton, although more work must be done to improve the accuracy of those estimations.     

Reproductive biology of a deep-sea brittle star Amphiura carchara (Echinodermata: Ophiuroidea)

 New information on the brooding reproduction of Amphiura carchara, and previous studies of several deep-sea congeners, contradict the prevailing notion that direct development is exceptional among deep-sea echinoderms. Over 500 specimens of A. carchara from 2,850 to 4,100-m depths off the coast of California, USA, were studied. The species was found to be gonochoric, although most brooding species of ophiuroids are hermaphroditic. Females each brooded up to 72 embryos at a time, with up to 10 in a single bursa. The embryos of individual adults were often at different stages of development, but those brooded in a single bursa tended to be at the same stage. Thus, the species has characteristics that breach the distinctions between sequential and simultaneous brooding exhibited by shallow-water ophiuroids. The embryos developed from yolky eggs that are large for an ophiuroid, with the mean largest oocytes 0.45 mm, and ranging to 1.28 mm in diameter. Almost all late-stage embryos were positioned with their mouth and arms pressed against the wall of the bursa, possibly to facilitate the uptake of nutrients from the parent. Thus, A. carchara may be matrotrophic. The largest embryos examined had a rudimentary disk skeleton, and arms with four joints and a terminal plate. Emerging juveniles probably differ in disk diameter. The species appears to brood year round, although differences in gonad size, the incidence of brooding, and the relative numbers of early developmental stages in summer and winter samples indicated that there are seasonal trends in reproduction. Received: 14 February 2000 / Accepted: 3 July 2000     

Identification of settling and early postlarval stages of mussels (Mytilus spp.) from the Pacific coast of North America, using prodissoconch morphology and genomic DNA

 Detailed inventories of the benthos and field studies of the settlement and recruitment processes of marine benthic invertebrates require accurate identification of newly settled larvae and early juvenile stages. We provide morphological criteria, visible under a good quality dissecting stereomicroscope, by which to discriminate between species of the settling larval and early postlarval stages (∼250 to 700 μm shell length) of mussels of the genus Mytilus on the west coast of Vancouver Island and Southern California. Compared to the bay mussel (M. trossulus), the sea mussel (M. californianus) has: (i) a shallower and flatter umbo, the latter corresponding to a significantly less pronounced prodissoconch I (PI) curvature and (ii) a greater PI length; as well as (iii) a wider separation between the provincular lateral teeth (PLT). The PLT distance is a new term denoting the separation between the midpoint of two reddish pigment spots of the provinculum (larval hinge apparatus) region of settling larvae and early postlarvae of Mytilus spp. from the East Pacific Coast. These spots mark the larger provincular lateral teeth, situated at either end of the provinculum. We confirmed the validity of morphological criteria by comparing PCR products of genomic DNA of provisionally identified postlarvae. Furthermore, measurements of PI lengths and PLT distance from well-preserved postlarvae of sea mussels (M. californianus) and of bay mussels (M. galloprovincialis) from Southern California indicate that the PI morphology and morphometry, and PLT distance criterion apply for that region as well. The criteria presented here can also apply to the advanced (competent) veliger stages, as the latter may settle (i.e. become the “settling” stage) upon encountering a suitable substrate. Our present and previously published work provide economical and effective identification methods that can be used to discriminate among early life history stages (∼250 μm to 5.0 mm shell length) of Mytilus spp. along the west coast of North America. Received: 10 November 1999 / Accepted: 6 September 2000     

环境DNA(eDNA)宏条形码技术对枝角类浮游动物物种鉴定及其生物量监测研究

环境DNA(eDNA)宏条形码(Metabarcoding)技术越来越多地被应用于环境中物种定性识别,但如何定量监测物种在环境中的丰度尚未得到解决。本研究以太湖流域常见的5种浮游动物拟同形溞、大型溞、蚤状溞、多刺裸腹溞、老年低额溞为研究对象,建立了一种基于eDNA宏条形码技术的物种定量方法,并与实时荧光定量PCR(qPCR)相比较,研究了eDNA宏条形码技术多物种定量的准确性。结果表明,PCR引物对eDNA宏条形码的物种检测和定量影响显著。313 bp COI313引物对浮游动物物种覆盖度高,但是物种间DNA扩增的偏好性大,不适用于eDNA宏条形码定量检测。基于COI序列重新设计的短COI116引物能够同时检测出所有5个物种。荧光定量PCR(qPCR)物种拷贝数与物种相对占比呈正相关。eDNA宏条形码所检出每个物种的序列数与qPCR定量拷贝数高度一致。综上,eDNA宏条形码技术可实现对浮游动物物种的半定量检测,在生物多样性监测和生物完整性评价有显著的应用价值。