Evolution of the tRNA gene family in mitochondrial genomes of five Meretrix clams (Bivalvia,Veneridae)

In contrast to the extreme conservation of nuclear-encoded tRNAs, organization of the mitochondrial (mt) tRNA gene family in invertebrates is highly dynamic and rapidly evolving. While gene duplication and loss, gene isomerism, recruitment, and rearrangements have occurred sporadically in several invertebrate lineages, little is known regarding the pattern of their evolution. Comparisons of invertebrate mt genomes at a generic level can be extremely helpful in investigating evolutionary patterns of variation, as intermediate stages of the process may be identified. Variation of mitochondrial tRNA organization among Meretrix clams provides good materials to investigate mt tRNA evolution. We characterized the complete mt genome of the lyrate Asiatic hard clam Meretrix lyrata, re-annotated tRNAs of four previously sequenced Meretrix clams, and undertook an intensive comparison of tRNA gene families in these clams. Our results 1) provide evidence that the commonly observed duplication of trnM may have occurred independently in different bivalve lineages and, based on the higher degree of trnM gene similarity, may have occurred more recently than expected; 2) suggest that “horizontal” evolution may have played an important role in tRNA gene family evolution based on frequent gene duplications and gene recruitment events; and 3) reveal the first case of isoacceptor “vertical” tRNA gene recruitment (VTGR) and present the first clear evidence that VTGR allows rapid evolution of tRNAs. We identify the trnS^− UCR gene in Meretrix clams, previously considered missing in this lineage, and speculate that trnS^− UCR lacking the D-arm in both M. lyrata and Meretrix lamarckii may represent the ancestral status. Phylogenetic analysis based on 13 concatenate protein-coding genes provided opportunities to detect rapidly evolved tRNA genes via VTGR and gene isomerism processes. This study suggests that evolution of the mt tRNA gene family in bivalves is more complex than previously thought and that comparison of several congeneric species is a useful strategy in investigating evolutionary patterns and dynamics of mt tRNA genes.     

Behavioral plasticity of the soft-shell clam, Mya arenaria (L.), in the presence of predators increases survival in the field

Soft-shell clams, Mya arenaria, are sessile, suspension-feeding bivalves that are preyed upon by the exotic green crab, Carcinus maenas. Clams evade crab consumers by burrowing deeper into the sediment after perceiving a threat from a nearby predator. The purpose of this study was to determine the types of signals that M. arenaria use to detect predators and the types of behaviors clams use to avoid being eaten. In a field study, clams increased their burial depth in the presence of green crab predators consuming conspecifics that were caged nearby, and also increased burial depth after artificial tactile stimulation in the laboratory assay. These results indicate that clams can use chemical and mechanical cues to detect potential predatory threats. We performed a field study to examine the difference in survivability of clams that had burrowed deeper into the sediment in response to predators vs. control clams that were burrowed less deeply. Significantly higher survival rates were observed in clams that had initially burrowed more deeply, suggesting that increasing burial depth is a valid predator avoidance strategy. Some bivalves also alter their pumping rates in the presence of predators, making them less apparent and providing more structural defense by covering soft tissue, and we measured pumping time of soft-shell clams in the presence and absence of predators, when burrowing was not an option for escape. Soft-shell clams did not alter their pumping time in the presence of green crab predators, possibly because they employ a burrowing method called “hydraulic” or “jet-propelled” burrowing, where it is necessary for the clam to pump in order to burrow. Chemical signals and tactile cues instigated behavioral changes in M. arenaria, and this change in behavior (increasing burial depth) increased clam survival in the field.     

Experimental test of biodeposition and ammonium excretion from blue mussels (Mytilus edulis) on eelgrass (Zostera marina) performance

Blue mussels and eelgrass have been found to coexist in many locations. However, knowledge of the interactions between these species is limited. Two experiments were conducted in the laboratory, a “Deposit” and an “Epiphyte” experiment. The Deposit experiment examined possible effects of increasing load of blue mussel (Mytilus edulis) biodeposits on sediment biogeochemistry and eelgrass (Zostera marina) performance. Z. marina mesocosms received normal or high loads of mussel biodeposits (Normal and High), while no biodeposits were added to the Control. High dosage had overall negative effects on Z. marina, which was reflected as lower leaf numbers and biomass and accumulation of elemental sulphur in rhizomes. The sediment biogeochemical conditions were altered, as the mussel biodeposits enhanced sulphate reduction rates and increased sulphide concentrations in the porewater, which may result in sulphide invasion and reduced growth of Z. marina.In the Epiphyte experiment effects of mussel excretion, with particular emphasis on ammonium, on the growth of Z. marina and their epiphytes were examined. A thick cover of epiphytes developed on Z. marina growing together with M. edulis, and the relative growth rate was reduced with 20% compared to plants from control without mussels. Overall the experiments showed negative effects on Z. marina growing together with M. edulis, thereby supporting a preceding field study by Vinther et al. [Vinther, H.F., Laursen, J.S., Holmer, M. 2008. Negative effects of blue mussel (Mytilus edulis) presence in eelgrass (Zostera marina) beds in Flensborg fjord, Denmark. Est. Coast Shelf. Sci. 77, 91-103.].     

Effects of coral transplantation and giant clam restocking on the structure of fish communities on degraded patch reefs

Active restoration is being practiced to supplement conservation activities for the purpose of reversing the trend of reef degradation. In the last decade, the feasibility of different restoration approaches such as coral transplantation and restocking of other marine biota has been the focus of research and relatively few have examined experimentally its effects on the resultant communities. In this study, coral transplantation and giant clam restocking were applied on 25 degraded patch reefs (~ 25 m²) inside a marine sanctuary in Pangasinan, northwestern Philippines to examine their effects on the community structure of reef fishes. Five interventions or treatments were employed: 1) “coral” consisted of transplantation of a combination of Acropora spp. and Pocillopora spp. on concrete blocks; 2) “clam” consisted of restocking of Tridacna gigas; 3) “clam+coral” consisted of restocking of T. gigas with Acropora spp. transplanted on their shells; 4) “shell” consisted of deployment of T. gigas shells; and 5) “control” consisted of no intervention. Fish communities on the patch reefs were monitored monthly for 3 months before the intervention and were monitored further for 11 months after the intervention, including 1 recruitment season. After the intervention, the coral cover and the “other biota” category increased in the coral and clam+coral treatments, due to the transplanted corals and deployed giant clams. Consequently, the complexity of the substrate was enhanced. A month after the intervention, a rapid increase in the abundance and species richness of reef fishes on the coral, clam+coral and clam treatments was observed compared to the shell and control treatments. A change in species composition of reef fish assemblage was also apparent in the coral and clam+coral treatments relative to the clam, shell and control, especially 4 months after the intervention. The present experiment demonstrates the feasibility of improving the condition of degraded patch reefs, which can subsequently enhance the fish community. Results also show the importance of the underlying substratum and the abundance of live corals and clams to reef fishes.     

Upper temperature tolerances of three molluscs from South African sandy beaches

The upper temperature tolerances of two South African bivalves, Donax serra Röding and D. sordidus Hanley, and a gastropod, Bullia rhodostoma (Reeve), from sandy beaches in Algoa Bay. were compared by means of median lethal temperatures (LT₅₀) and median burial temperature (BT₅₀) determinations for periods of exposure up to 96 h. Donax serra and D. sordidus adults showed a similar temperature tolerance of 29°C. D. serra juveniles showed a lower tolerance of 27°C. Bullia rhodostoma had a slightly higher thermal tolerance (≈ 31°C) than the bivalves, with small individuals having a greater thermal tolerance than large individuals for the longer exposure periods. These thermal tolerances are discussed in relation to distribution, and compared with those of related species from European waters.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

Analysis of protein aggregation kinetics using short amino acid peptide tags

Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10 mg/ml to 25.0 mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the “transient solubility (TS)”, the “aggregation initiation concentration (AIC)” and the “long-term solubility (LS)”. AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the “solubility” of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein.     

Hypoxia and sulphide influence gamete production in Ulva sp.

Gamete production after exposure to hypoxia or sulphide was studied in the marine macroalga Ulva sp. collected in the Sacca di Goro, Italy. Experiments were carried out on discs (12 mm diameter) of thalli cultured in artificial sea water in laboratory at 20 ± 1 °C, 152 μmol m⁻² s⁻¹, 16 h photoperiod and 30‰ salinity. Dehydration of thallus was used as inducer of gametogenesis and growth and gamete release during recovery after 10, 20, 30 or 40 min dehydration (20 ± 1 °C, 25% humidity) were analysed. Unlike non-dehydrated thalli the dehydrated ones produced gametes. Thallus discs, non-dehydrated or subjected to 30 min dehydration, were exposed to hypoxia (1.78–4.02 μmol O₂ L⁻¹) or sulphide (1 mM) for 3, 5, or 7 days at 20 °C in the dark. Non-dehydrated and dehydrated thalli maintained in normoxic conditions in the dark were the controls. Gamete density was checked by counting at the end of the incubation period and during the subsequent 7 days of recovery under 16 h photoperiod in normoxic conditions. Non-dehydrated thalli maintained in normoxic conditions in the dark released gametes when returned to light suggesting that dark constitutes a stimulus to gamete production. The presence of gametes at the end of 3 days incubation of dehydrated thalli in normoxia demonstrated that gametogenesis can occur even in the dark. However, gametes were not present at the end of incubation in hypoxic and sulphidic conditions. Actually, during hypoxic incubation oxygen consumption in D-thalli was very low, only 0.117 × 10⁻³ μmol O₂ mg⁻¹ h⁻¹ compared to 5.93 × 10⁻³ μmol O₂ mg⁻¹ h⁻¹ in normoxia, denoting a reduction of the metabolic rate that could not sustain gametogenesis. During recovery after incubation in normoxic, hypoxic or sulphidic conditions densities of gametes from dehydrated thalli showed significant differences and resulted after hypoxia > after normoxia > after sulphide. Differences in non-dehydrated thalli were not significant. Dehydrated thalli, still green at the end of the incubation period, underwent blanching in the course of recovery in parallel to gamete production, while non-dehydrated thalli maintained their green colour even after exposure to sulphide. Our findings suggest that macroalga Ulva sp. can survive exposure to darkness, severe hypoxia and high sulphide levels and can maintain gamete production even when the exposure to these stress conditions is joined to dehydration.     

Molecular cloning and functional analyses of glutathione peroxidase homologous genes from Chlorella sp. NJ-18

The extreme variability of the mitochondrial (mito) genomes of bivalves makes it difficult to understand their evolutionary dynamics, given that species from different families do not share comparable features. We compared the mitogenomes from four Paphia clams (three of them were firstly sequenced) and found that mitogenome reorganization among the four congeneric species is not random but follows phylogenetic trends. Start/stop codon variations are species-correlated rather than gene-correlated, and bear useful phylogenetic information. Unique start/stop codon usage in P. euglypta and A+T content in P. amabilis indicates that these mitogenome-level characters, usually considered to be conservative features in other lineages, may not be phylogenetically evolved, but may have evolved via species-specific mitogenomic maintenance mechanisms. Variable divergence of two trnM genes in different lineages may demonstrate differences in mechanisms by which paralogous trnM genes are maintained. Sequence alignment analysis indicates that the VNTRs in the four mitogenomes have a common origin. The rationale of the subgenus Neotapes Kuroda and Habe, 1971 was supported by evidence from morphological characters, mitogenomic features, as well as phylogenetic analyses using cox1 and rrnS genes. The data suggest that the taxonomic basis of the subgenus should be “smooth surface” but not “undulated lines,” and P. textile should be classified to the Neotapes subgenus.     

Burrowing behaviour of the softshell clam (Mya arenaria) following erosion and transport

Erosion and transport of juvenile individuals may alter the distribution pattern of intertidal bivalves. The burrowing success of recently transported juvenile softshell clams (Mya arenaria) was studied in a laboratory flume under a wide range of hydrosedimentary environments. Juvenile individuals (5-20 mm) were observed under a simulated 30 min slack tide before initiating the flow for a period of 60 min. Five different free-stream velocities (0, 3, 5, 10 and 24 cm s^− 1) and four sediment types (mud, sandy-mud, sand and gravel) were used. The mean proportion of juvenile clams that initiated (MPI) or completed (MPC) a burial decreased with increasing shell length. Erosion from the sediment was more important in large juveniles suggesting that large juveniles may have more difficulty successfully relocating once transported. The MPI increased with increasing flow speed in experimental runs held at speed < 24 cm s^− 1. This was observed in all sediment types. Most individuals were unable to burrow at 24 cm s^− 1 because they got eroded. The MPC also increased with increasing flow speed in mud, sandy-mud and sand. The MPC's response to flow was more complex in gravel because of a shell length × flow speed interaction effect. Our observations suggest that water movement may induce the burrowing behaviour of recently eroded juvenile clams. Results are discussed in an ecological and aquacultural context.     

The influence of plankton composition and water quality on hard clam (Mercenaria mercenaria L.) populations across Long Island's south shore lagoon estuaries (New York, USA)

We conducted a two-year study to assess how plankton composition and water quality impacts the distribution, densities, condition, growth, biochemical composition and reproductive success of juvenile and adult Mercenaria mercenaria (L.) in Long Island's south shore estuaries (LISSE). Juvenile and adult hard clams were placed in suspended cages at 10 locations ranging from the ocean inlets to locations furthest from inlets in Shinnecock Bay (SB), the eastern-most barrier island estuary of LISSE, and Great South Bay (GSB), the western-most barrier island estuary of LISSE. Phytoplankton community composition, temperature, salinity, dissolved oxygen, and clam growth and condition were monitored bi-weekly. A benthic survey of M. mercenaria densities in both estuaries was also conducted. In both 2004 and 2005, juveniles in central bay locations had significantly faster growth rates, lower mortality rates, and higher lipid content relative to sites closest to the inlets. Adult hard clams closest to the Fire Island inlet also had significantly lower condition indexes compared to mid-bay stations and densities of wild M. mercenaria populations in both estuaries were lower near inlets compared to locations further from inlets. In addition to substantial spatial differences within each estuary, differences were also observed between the embayments as juvenile clams in SB grew approximately twice as fast as those in GSB and adults in SB had significantly greater condition indexes than clams in GSB. Instantaneous juvenile growth rates were highly correlated to temperatures below 24 °C (p < 0.0001) and were also significantly correlated with several indicators of suspended food quantity and food quality (centric diatoms, phytoplankton cells > 5 μm, and dinoflagellates (inverse correlation)) which co-varied independently of temperature. In sum, these results suggest tidal exchange in LISSE promotes a water quality regime (cold water, with low food concentration) which would reduce the growth of juvenile clams and the overall reproductive success of adult hard clams located near newly-formed ocean inlets. However, increased exchange for regions furthest from inlets could enhance juvenile clam growth rates by reducing summer peak temperatures (> 24 °C) and densities of poor food sources (dinoflagellates).     

Molluscan mobile elements similar to the vertebrate Recombination-Activating Genes

Animal genomes contain ∼20,000 genes. Additionally millions of genes for antigen receptors are generated in cells of the immune system from the sets of separate gene segments by a mechanism known as the V(D)J somatic recombination. The components of the V(D)J recombination system, Recombination-Activating Gene proteins (RAG1 and RAG2) and recombination signal sequence (RSS), are thought to have “entered” the vertebrate genome as a hypothetical “RAG transposon”. Recently discovered mobile elements have terminal inverted repeats (TIRs) similar to RSS and may encode proteins with a different degree of similarity to RAG1. We describe a novel N-RAG-TP transposon identified from the sea slug Aplysia californica that encodes a protein similar to the N-terminal part of RAG1 in vertebrates. This refines the “RAG transposon” hypothesis and allows us to propose a scenario for V(D)J recombination machinery evolution from a relic transposon related to the existing mobile elements N-RAG-TP, Chapaev, and Transib.     

Leaf essential oil composition of 10 species of Ocotea (Lauraceae) from Monteverde,Costa Rica

The leaf essential oils of 10 species of Ocotea (Lauraceae) from Monteverde, Costa Rica (Ocotea floribunda, Ocotea holdridgeana, Ocotea meziana, Ocotea sinuata, Ocotea tonduzii, Ocotea valeriana, Ocotea veraguensis, Ocotea whitei, and two undescribed species, Ocotea new species “los llanos”, and Ocotea new species “small leaf”) have been obtained by hydrodistillation and analyzed by GC–MS in order to discern the differences and similarities between the volatile chemical compositions of these species. The principal common constituents of the 10 species of Ocotea were α-pinene, β-pinene, β-caryophyllene, and germacrene-D.     

Sequential binding of FurA from Anabaena sp. PCC 7120 to iron boxes: Exploring regulation at the nanoscale

Fur (ferric uptake regulator) proteins are involved in the control of a variety of processes in most prokaryotes. Although it is assumed that this regulator binds its DNA targets as a dimer, the way in which this interaction occurs remains unknown. We have focused on FurA from the cyanobacterium Anabaena sp. PCC 7120. To assess the molecular mechanism by which FurA specifically binds to “iron boxes” in P_furA, we examined the topology arrangement of FurA–DNA complexes by atomic force microscopy. Interestingly, FurA–P_furA complexes exhibit several populations, in which one is the predominant and depends clearly on the regulator/promoter ratio on the environment. Those results together with EMSA and other techniques suggest that FurA binds P_furA using a sequential mechanism: (i) a monomer specifically binds to an “iron box” and bends P_furA; (ii) two situations may occur, that a second FurA monomer covers the free “iron box" or that joins to the previously used forming a dimer which would maintain the DNA kinked; (iii) trimerization in which the DNA is unbent; and (iv) finally undergoes a tetramerization; the next coming molecules cover the DNA strands unspecifically. In summary, the bending appears when an “iron box” is bound to one or two molecules and decreases when both “iron boxes” are covered. These results suggest that DNA bending contributes at the first steps of FurA repression promoting the recruitment of new molecules resulting in a fine regulation in the Fur-dependent cluster associated genes.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

Merychippus (Mammalia, Perissodactyla, Equidae) from the Middle Miocene of state of Oaxaca, southeastern Mexico

Mexican material referable to Merychippus from two localities in eastern Oaxaca was described first nearly 50 years ago. Subsequent work there and in Central Oaxaca, spanning some 30 years, has allowed to establish the detail stratigraphy in both regions, and assembled a collection of merychippine material from the Matatlán (Central Oaxaca) and El Camarón (eastern Oaxaca) Formations, both K-Ar dated ~15 Ma (late early Barstovian). Detailed taxonomic analysis of this collection indicate the presence of two subhypsodont horse species referable to “Merychippus” cf. “M.” primus and “M.” cf. “M.” sejunctus in both regions. These records document the coexistence in tropical southern North America of basal and hipparionine affinity merychippine grade species, and provide a glimpse in to the diversity of subhypsodont equids in this region.