Eggs of the copepod Acartia tonsa Dana require hypoxic conditions to tolerate prolonged embryonic development arrest.

BACKGROUND: Copepods make up the largest zooplankton biomass in coastal areas and estuaries and are pivotal for the normal development of fish larva of countless species. During spring in neritic boreal waters, the copepod pelagic biomass increases rapidly from near absence during winter. In the calanoid species Acartia tonsa, a small fraction of eggs are dormant regardless of external conditions and this has been hypothesized to be crucial for sediment egg banks and for the rapid biomass increase during spring. Other eggs can enter a state of induced arrest called quiescence when external conditions are unfavourable. While temperature is known to be a pivotal factor in the transition from developing to resting eggs and back, the role of pH and free Oxygen in embryo development has not been systematically investigated. RESULTS: Here, we show in a laboratory setting that hypoxic conditions are necessary for resting eggs to maintain a near-intact rate of survival after several months of induced resting. We further investigate the influence of pH that is realistic for natural sediments on the viability of resting eggs and document the effect that eggs have on the pH of the surrounding environment. We find that resting eggs acidify their immediate surroundings and are able to survive in a wide range of pH. CONCLUSIONS: This is the first study to demonstrate the importance of hypoxia on the survival capabilities of A. tonsa resting eggs in a controlled laboratory setting, and the first to show that the large majority of quiescent eggs are able to tolerate prolonged resting. These findings have large implications for the understanding of the recruitment of copepods from sediment egg banks, which are considered the primary contributor of nauplii seeded to pelagic populations in nearshore habitats in late spring.

Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax.

The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active at concentrations above 270 µmol.

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax.

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm , of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.

Hyperactive antifreeze proteins from longhorn beetles: some structural insights.

This study reports on structural characteristics of hyperactive antifreeze proteins (AFPs) from two species of longhorn beetles. In Rhagium mordax, eight unique mRNAs coding for five different mature AFPs were identified from cold-hardy individuals. These AFPs are apparently homologues to a previously characterized AFP from the closely related species Rhagium inquisitor, and consist of six identifiable repeats of a putative ice binding motif TxTxTxT spaced irregularly apart by segments varying in length from 13 to 20 residues. Circular dichroism spectra show that the AFPs from both species have a high content of ß-sheet and low levels of α-helix and random coil. Theoretical predictions of residue-specific secondary structure locate these ß-sheets within the putative ice-binding motifs and the central parts of the segments separating them, consistent with an overall ß-helical structure with the ice-binding motifs stacked in a ß-sheet on one side of the coil. Molecular dynamics models based on these findings show that these AFPs would be energetically stable in a ß-helical conformation.