Single-step species identification of bivalve larvae using multiplex polymerase chain reaction

One of the biggest obstacles to studying recruitment variation in marine bivalves is the need to collect and process large numbers of plankton samples. Larval bivalves are notoriously difficult, if not impossible, to identify to species using morphological criteria alone. Remote time-series collections could satisfy the sampling challenge, but efficient identification techniques must be developed to obtain species-specific data. Thus, we have developed a multiplex polymerase chain reaction (PCR) identification assay in which a single reaction is capable of accurate and efficient discrimination of five target bivalve species based on the size of cytochrome oxidase I products. The assay was tested with cultured and field-sampled larvae as well as adult genomic DNAs. Using a single whole larva as template, multiplex PCR reactions were capable of discriminating among the commercially important bivalves: Mercenaria mercenaria, Argopecten irradians, Mulinia lateralis, Spisula solidissima and Mya arenaria. Overall accuracy was 92%, including very few false positives. The efficiency of this assay stems from its ability to discriminate multiple target species with a single molecular step that ultimately can be automated to process large numbers of larvae. Received: 30 March 2000 / Accepted: 10 July 2000     

三种纳米金颗粒对聚合酶链式反应扩增效率和特异性的影响

比较了采用柠檬酸三钠(Trisodium citrate,Na3Ct)还原法、聚乙烯吡咯烷酮(Polyvinylpyrrolidone,PVP)包裹法和巯基丙酸-同型半胱氨酸(Mercaptopropionic Acid-Homocystine,MPA-HCys)包裹法制备的3种纳米金颗粒(Gold nanoparticles,AuNPs)对聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增效率及特异性的影响,以评估金纳米材料表面性质与DNA生物大分子之间的相互作用.实验结果显示,3种AuNPs均能显著提高PCR效率,它们提高PCR效率的能力依次为:Na3 Ct-AuNPs(12 nm)MPA-HCys-AuNPs(12 nm)PVP-AuNPs(4 nm),而且这种增强效应与其浓度和尺寸相关.相同实验条件下,具有相似尺寸大小的Na3Ct-AuNPs比MPA-HCys-AuNPs更能有效提高PCR效率,这说明AuNPs的表面性质也是影响PCR效率的重要因素之一.另外,3种AuNPs均能有效消除竞争性引物对PCR的抑制作用,并且这种作用与其浓度密切相关.上述研究结果表明,具有不同表面性质的3种AuNPs均能有效提高PCR效率和特异性,并且这种作用与其浓度、尺寸和表面性质密切相关.     

Acclimation of the European sea bass to freshwater: monitoring genetic changes by RAPD polymerase chain reaction to detect DNA polymorphisms

We investigated the use of the polymerase chain reaction (PCR) and the associated random amplification of polymorphic DNA (RAPD) technique to study variation in samples of the sea bass, Dicentrarchus labrax, before and after acclimation to freshwater. Acclimation trials were repeated twice, in 1989 and 1990, for two samples originating from the same broodstock (individuals from different localities along the Italian coasts), with overall mortality rates averaging 94 and 75% in the 2 yr, respectively. Analyses are based on 126 polymorphic RAPD markers, scored in at least 39 individuals for each of the starting and acclimated samples in both years. Analysis of RAPD patterns revealed high levels of DNA polymorphism in both 1989 and 1990 starting samples. The acclimated samples maintained similar polymorphism levels. Shifts in marker frequencies between starting and acclimated samples occurred in both years. A correspondence analysis carried out on multimarker individual profiles suggests that the two starting samples resulted from uneven sampling of the same heterogenous broodstock. This analysis clearly separates RAPD phenotypes from starting and acclimated samples in both years and identifies the RAPD markers responsible for such displacement. Patterns of RAPD variation are compared with previous allozymic studies carried out on the same samples. A major difference between the two studies was the number of markers available. Fewer allozyme loci were studied than were RAPD markers. The cause of repeated shifts for allozyme alleles in replicate experiments were almost certainly due to selection, while statistical chance could explain the repeated shift of only one out of more than 100 RAPD markers. We have shown that RAPD analysis, if carried out carefully, is quite reproducible and sensitive enough to reveal high levels of variation among individuals from the same broodstock. A major drawback of this approach is the still unclear inheritance patterns of RAPD polymorphisms. The use of multivariate analyses is suggested as a possible alternative to traditional population genetics techniques to analyze patterns of variation in the absence of a precise genetic interpretation.     

A molecular method for the identification of resting eggs of acartiid copepods in the Thau lagoon, France

Acartia and Paracartia species, often known to co-occur, can exhibit complex life cycles, including the production of resting eggs. Studying and understanding their population dynamics is hindered by the inability to identify eggs and early developmental stages using morphological techniques. We have developed a simple molecular technique to distinguish between the three species of the Acartiidae family (Acartia clausi, A. discaudata and Paracartia grani) that co-occur in the Thau lagoon (43°25′N; 03°40′E) in southern France. Direct amplification of a partial region of the mitochondrial cytochrome oxidase I gene by polymerase chain reaction and subsequent restriction fragment length polymorphism results in a unique restriction profile for each species. The technique is capable of determining the identity of individual eggs, including resting eggs retrieved from sediment samples, illustrating its application in facilitating population dynamic studies of this ubiquitous and important member of the zooplankton community.     

Mesozooplankton resting eggs in the Baltic Sea: identification and vertical distribution in laminated and mixed sediments

Benthic resting eggs of rotifers, cladocerans and calanoid copepods were studied at two sites with different hydrographical and sediment properties off the SW coast of Finland, the northern Baltic Sea between 1991 and 1993. Vertical distribution of the resting eggs in the sediment was studied down to 10 cm depth, and hatching of the eggs extracted from different 1-cm thick sediment layers was experimentally tested. The larvae of eight mesozooplankton taxa emerged from the eggs: Calanoida (mostlyAcartia bifilosa), cladoceransBosmina spp.,Daphnia spp.,Evadne nordmanni, andPodon spp. (mostlyP. polyphemoides), and rotifersSynchaeta spp.,Keratella spp. andAsplanchna priodonta. Some calanoid and cladoceran eggs hatched after 7 mo storage of the sediment at 3°C. A few calanoid and cladoceran eggs hatched from the 8 to 10 cm deep sediment layer. Their age, estimated by means of¹³⁷Cs analysis, was ca. 8 to 10 yr. Vertical distribution of the benthic eggs reflected the different sediment structures of the study sites. The eggs of all zooplankton taxa were more stratified in the laminated sediment than in the disturbed sediment. We suggest that the intensity of deep water mixing is the key factor for regulating the vertical distribution and hatching probability of mesozooplankton resting eggs, since it largely determines the stratification of the sedimenting material, distribution of oxygen within the sediment, and the living conditions of benthic animals causing bioturbation.     

A density-gradient column for determining the specific gravity of fish eggs,with particular reference to eggs of the mackerel Scomber scombrus

A new method is described for determining the specific gravity of fish eggs. The technique requires the preparation of a stable column of a continuously graded solution of sea-water salts such that a specimen introduced into the column settles at a level of known specific gravity, where it is in hydrostatic equilibrium with the fluid in the column. The method allows the simultaneous measurement of specific gravity of a number of specimens continuously throughout development and is suitable for use aboard a vessel at sea. Differences of specific gravity may be resolved to a precision of better than 4x10^-5; as an example of use of the method, the specific gravity of eggs of the mackerel Scomber scombrus L. declined during development within the range 1.0205 to 1.0245; unfertilized eggs displayed a continuous increase of specific gravity.     

Energy substrates for eggs and prefeeding larvae of the dolphinCoryphaena hippurus

Changes in the chemical composition of developing dolphin (Coryphaena hippurus) eggs and prefeeding yolksac larvae were determined in order to estimate probable dietary requirements of first-feeding larvae. Daily dry matter, protein nitrogen (PN), non-protein nitrogen (NPN), lipid, gross energy content, fatty acid and amino acid profiles from Day 1 to Day 2 eggs and Day 1 to Day 3 larvae were compared. Lipid was the primary endogenous energy source accounting for the daily caloric deficit through both the egg and larval stages, except over the day of hatching. The catabolism of lipid by embryos (0.078 cal d^–1) was greater than that by yolksac larvae (0.036 cal d^–1). The higher demand for energy by embryos was related to a greater rate of protein synthesis during the egg stage. The ratio of PN:NPN increased during egg development without change in total nitrogen content, but was constant throughout the yolksac larvae period. The lipid content per embryo did not decrease over the hatching period (Day 2 to 3, postspawning). However, there was a loss in amino acid content not totally accounted for by sloughing of the chorion at hatching. This loss, as protein, accounted for 0.053 cal of gross energy, which represented 70% of the total estimated energy needs of the fish over this period. Loss of non-essential amino acids (25%) was higher than that of essential amino acids (13%). Proline and tyrosine accounted for 32% of the total loss of amino acids at this time. The only preferential use of fatty acids over any period was a small but significant drop in the content of C22:6n-3 prior to the onset of feeding (Day 5, postspawning). It is speculated that the pattern of energy-substrate use of first-feeding dolphin larvae will reflect the pattern of endogenous energy use during the egg and prefeeding yolksac larval stages. Diets or feeding regimens with lipid as the primary energy source, and containing a fatty acid profile similar to that of eggs or yolksac larvae, should be useful in culturing this species, at least during the early feeding stages.     

Respiration of the eggs of the giant cuttlefish Sepia apama

On the roofs of subtidal crevices, the giant cuttlefish (Sepia apama) of southern Australia lays clutches of lemon-shaped eggs which hatch after 3 to 5 mo. Diffusion of oxygen through the capsule and chorion membrane to the perivitelline fluid and embryo was modelled using the equation V˙ _O2 = G _O2(P _O2out−P _O2in), where V˙ _O2 = rate of oxygen consumption, G _O2 = oxygen conductance of the capsule, and P _O2 values = oxygen partial pressures across the capsule. During development, V˙ _O2 rose exponentially as the embryo grew, reaching 5.5 μl h⁻¹ at hatching. Throughout development, the capsule dimensions enlarged by absorption of water into the perivitelline space, increasing G _O2 by a combination of increasing surface area, and decreasing thickness of the capsule. These processes maintained P _O2in high enough to allow unrestricted V˙ _O2 until shortly before hatching. Diffusion limitation of respiration in hatching-stage embryos was demonstrated by (1) increased embryonic V˙ _O2 when P _O2out was experimentally raised, (2) greater V˙ _O2 of resting individuals immediately after hatching, and (3) reduced V˙ _O2 of hatchlings at experimental P _O2 levels higher than P _O2in before hatching. Thus, low P _O2in may be the stimulus to hatch. Potential problems of diffusive gas-exchange are mitigated by the relatively low incubation temperature (12 °C), which may be a factor limiting the distribution of the species to cool, southern waters. Received: 14 August 1999 / Accepted: 24 January 2000     

Osmotic properties of eggs of the herring Clupea harengus

Yolk osmolality of developing eggs of the herring Clupea harengus L. is strongly hypoosmotic to seawater: about 440 mOsm during the first week of development decreasing to 360 mOsm before hatching. The perivitelline fluid (PVF) of the eggs is isoosmotic to the ambient medium. The PVF equilibrates within 10 min to changes in the ambient seawater. The content of Na⁺, K⁺, Cl^- and free amino acids amount to 26, 52, 48, and 54 n mol egg^-1, respectively, on Days 1 to 3 after fertilization, increasing to 63, 69, 80, and 79 n mol egg^-1, respectively, prior to hatching (Days 18–20). The apparently conflicting findings of a decreasing yolk osmolality and a simultaneous increase in the amount of egg solutes are at present unexplained.     

Calanoid copepod eggs in sea-bottom muds. IV. Effects of some environmental factors on the hatching of resting eggs

During cadmium uptake by the shore crab Carcinus maenas (L.), cadmium appears in both haemolymph and urine within 1 h, and comes into equilibrium with the haemolymp after some 60 h. The equilibrium concentration for haemolymph cadmium is always considerably less than that of the external medium, whether the latter is close to background level or at the artificially high concentration of 2.2 mg l^-1. Despite earlier evidence that cadmium entering the haemolymph is mainly bound to protein, current results show most of this to be freely labile and quickly displaced by cadmium taken up subsequently. There is some evidence to suggest that cadmium is passed on from the haemolymph to the hepatopancreas. A large proportion of whole-body cadmium is adsorbed onto the exoskeleton and gills. The comparatively rapid efflux of ¹⁰⁹Cd from loaded crabs over a period of 1 week is mainly due to desorption of this cadmium from the outer body surface.     

航道疏浚泥悬浮液对黑鲷胚胎的发育及仔鱼摄食生长的影响

采用静水试验法,温度在20℃左右,并保持试验用水溶解氧为饱和溶氧的60%,用疏浚泥悬浮液与过滤海水按等对数间距稀释成4个体积浓度水平(1:2、1:10、1:50、1:200),同时以过滤海水为对照组,每一个浓度组设3个平行样本,对人工养殖的黑鲷Spaptls macrocephalus鱼卵和仔鱼进行毒性试验.试验结果表明:疏浚泥悬浮液对鱼卵孵化有一定影响,但不显著.仔鱼的全长和体重的增长与悬浮液浓度的变化并无十分明显的对应关系,只有当水体中的悬浮液达到一定浓度时,才显现出对仔鱼生长的影响作用.在仔鱼摄食过程中,悬浮液对全长生长产生的抑制或影响作用不显著,但高浓度的悬浮液则对体重增长产生了一定的抑制性,不过这是一种短暂性的影响,随着时间的延长,其作用逐渐减弱或消失.     

The use of legumes for reclaiming copper mine wastes in the Southwestern USA

Seeds of three legumes, blue lupine (Lupinus hirsutus L.), sesbania (Sesbania macrocarpa Muhl.), and alfalfa (Medicago sativa L.) were broadcast planted on four soil materials (desert soil, overburden, overburden plus tailings, and tailings) associated with copper mines near Tucson, Arizona. Experiments were conducted to study the effects of the four soil materials on germination (emergence), seedling establishment, and growth over a two-year period (1974 and 1975). All species grew best on desert soil, followed by overburden, overburden plus tailings, and tailings, in decreasing order. The presence of copper in overburden plus tailings and in tailings may have created a toxicity effect that limited the growth of plants in these two soil materials. Alfalfa was superior on all soil materials. Sesbania and blue lupine grew equally well. All three species were able to adapt to the different soil materials. For revegetation on copper mine wastes, it is desirable to plant a variety of plant species rather than a single species, to help blend the disturbed areas into the surrounding environmentApproved for publication as Arizona Agricultural Experiment Station Research Contribution No. 3100.     

Paternity control for externally fertilised eggs: behavioural mechanisms in the waterfrog species complex

Male competition for mates and female mate choice are key mechanisms involved in sexual selection. Surprisingly, these mechanisms have often been investigated separately although they appear to interact in many species. Male–male competition for territories located at the best places or to establish dominance relationships often explain mating patterns. Such male behaviours may affect and sometimes even hinder female mate choice, as in the case of sexual coercion. While in many species females are able to exert cryptic control over paternity (i.e. a process allowing females to bias offspring production toward certain males after intromission), in other species external fertilisation prevents females from doing so. This is the case in the waterfrog hybridisation complex where the hybrid Pelophylax esculentus can only produce viable offspring by pairing with the parental species Pelophylax lessonae (hybridogenetic reproduction). We examined two potential processes that could enhance such mating combinations. Firstly, by monitoring male spatial distribution within six choruses, we showed that the proportion of P. lessonae males located at the edge (in the best position to grasp females arriving at the chorus) cannot explain the frequency of mating combinations observed. Secondly, an experimental approach emphasised a new way for anuran females to favour paternity of a particular male in a sexual coercion context. When females are forcefully paired with an incompatible male, they cannot remove the male grasped on their back by themselves. Nevertheless, by controlling the movement of the pair within the chorus, these females often change mates by enhancing male competition instead of laying eggs. In many species with externally fertilised eggs, it may be thus necessary to take into account this new possibility for females to control offspring paternity.     

The use of metabolic ratios for determining the catabolic substrates of a solitary ascidian

In a previous study, Hatcher (1989: Mar. Biol. 102: 445–452) found that variations in CO₂ and O₂ respiration rates in individual marine invertebrates led to RQ (respiratory quotient) values which were variable and often outside theoretical limits. The present study was designed to examine the variability in several excretionbased metabolic ratios which are often used as alternatives for the RQ in qualitative predictions of catabolic substrates. The experimental organism was a solitary ascidian,Herdmania momus (Savigny), collected near Perth, Western Australia, between January and July 1984. Respiration and excretion rates ofH. momus were examined as a function of a progressive nutritional stress, and covariation was examined. Nutritional stress accounted for more of the variation over time in respiration rates (40 to 50%) than in NH ₄ ⁺ excretion rates (20%). Significant net exchanges of dissolved organic compounds were measured. Qualitative predictions of catabolic substrates were based on a comparison of metabolic ratios with theoretical limits. The O:PO ₄ ^3- ratios were lower than the theoretical limits. The values of the O:NH ₄ ⁺ and NH ₄ ⁺ :PO ₄ ^3- ratios were not influenced by nutritional conditions but changed as a function of reproductive condition of the ascidians. Based on the results of this study, it was concluded that metabolic ratios measured on individual ascidians cannot be reliable predictors of catabolic substrates.     

The use of simulation modeling to evaluate the mechanisms responsible for the nutritional benefits of food-for-protection mutualisms

In some mutualisms, a plant or insect provides a food resource in exchange for protection from herbivores, competitors or predators. This food resource can benefit the consumer, but the relative importance of different mechanisms responsible for this benefit is unclear. We used a colony-level simulation model to test the relative importance of increased larval production, increased worker foraging and increased worker survival for colony growth of fire ants, Solenopsis invicta, that consume plant-based foods. Increased food for larvae had the largest effect on colony growth of S. invicta followed by decreased worker mortality. Increased foraging rate had a small effect in the simulation model but data from a small laboratory experiment and another published study suggest that plant-based foods have little or no effect on foraging rates of S. invicta. Colony growth steadily increased the longer plant-based food was available and colonies were most responsive to plant-based food in the early summer (i.e., June). Our results demonstrate that population level simulation modeling can be a useful tool for examining the ecology of mutualistic interactions and the mechanisms through which species interact.     

Laboratory rearing of the clupeid fish Harengula pensacolae from fertilized eggs

Pelagic eggs of the scaled sardine Harengula pensacolae (Goode and Bean), have been hatched and reared in the laboratory for the first time. Larvae were reared in two 75 l aquaria under constant illumination, at an average temperature of 26.2°C. Zooplankton collected in a 35 mesh net was fed to the newly hatched larvae, and the diet was supplemented later with Artemia salina nauplii and a pelleted food. Larvae hatched at 4 mm TL (total length), and metamorphosed about 25 days later at 25 to 30 mm TL. Survivors averaged 76 mm TL 100 days after hatching. Of the 500 incubated eggs, 2.8% survived until 20 days, after which no significant natural mortality occurred. Sources of natural mortality included starvation, a copepod parasite (Caligus sp.), and injuries from contact with the sides of the tank. Larvae began feeding at 4.5 mm TL on copepod nauplii averaging 62 in body width. Scaled sardines were photopositive throughout the larval stage.Contribution No. 149, Bureau of Commercial Fisheries Tropical Atlantic Biological Laboratory, Miami, Florida 33149, USA.     

An adaptive annual rhythm in the sex of first pigeon eggs

When the reproductive value of male and female offspring varies differentially, parents are predicted to adjust the sex ratio of their offspring to maximize their fitness (Trivers and Willard, Science 179:90–92, 1973). Two factors have been repeatedly linked to skews in avian offspring sex ratio. First, laying date can affect offspring sex ratio when the sexes differ in age of first reproduction, such that the more slowly maturing sex is overproduced early in the season. Second, position of the egg in the laying sequence of a clutch may affect sex ratio bias since manipulating the sex of the first eggs may be least costly to the mother. We studied both factors in two non-domesticated pigeon species. Both the Wood pigeon (Columba palumbus) and the Rock pigeon (Columba livia) have long breeding seasons and lay two-egg clutches. In the field, we determined the sex of Wood pigeon nestlings. In Rock pigeons, housed in captivity outdoors, we determined embryo sex after 3 days of incubation. On the basis of their sex-specific age of first reproduction, we predicted that males, maturing at older age than females, should be produced in majority early and females later in the year. This was confirmed for both species. The bias was restricted to first eggs. Rock pigeons produced clutches throughout the year and show that the sex of the first egg followed an annual cycle. To our knowledge, this study presents the first evidence of a full annual rhythm in adaptive sex allocation in birds. We suggest that this reflects an endogenous seasonal program in primary sex ratio controlled by a preovulatory mechanism.     

Specificity and sensitivity of microsatellite markers for the identification of larvae

The identification of larval marine invertebrates to species or even higher taxonomic levels by morphological examination is notoriously difficult. Many diagnostic features are absent or poorly formed at early stages in development. This is particularly true for the larvae of bivalve molluscs, for which a routine and accurate method of identification would prove valuable to both ecologists and fishery managers. A simple molecular genetic method to identify specifically larvae of the European oyster, Ostrea edulis L., 1758, is presented. The test is based on PCR amplification of highly species-specific microsatellite loci and is sensitive enough to register the presence of a single larval individual (~200 µm width) in a mixed sample of 20 mg wet weight plankton (approximately 250 larval animals). This work demonstrates that microsatellite loci can be used as highly sensitive and specific taxonomic indicators, for studies of planktonic larvae. Details of three novel microsatellite loci are also given for O. edulis, increasing the suite of molecular tools available for use in population genetic studies of this commercially important species.     

The use of ICP-MS for the analysis of natural waters and an evaluation of sampling techniques

Analytical and sampling methods for a reconnaissance survey of river waters in former metal mining areas of Wales, UK, are considered. A semi-quantitative mode of analysis with a single internal standard, possible by inductively coupled plasma mass spectrometry (ICP-MS) is compared with more conventional quantitative ICP-MS methods using external calibration from aqueous standards. Semi-quantitative analysis can be much quicker than a more rigorous quantitative approach and gives values which agree with fully quantitative data to within 30%, indeed, agreement is often much better than this. A comparison of samples filtered and acidified in the field with untreated samples is also presented. This shows that in most cases it is unnecessary to filter and acidify samples if the storage time before analysis is short (in this case typically less than four days).