Biological weighting functions for DNA damage in sea urchin embryos exposed to ultraviolet radiation

Laboratory experiments examining the effects of ultraviolet radiation (UVR, 290-400 nm) on DNA damage were carried out using the embryos of three species of sea urchins from different habitats; Strongylocentrotus droebachiensis from the Gulf of Maine, Sterechinus neumayeri from the Antarctic, and Evechinus chloroticus from New Zealand. All three species exhibited significant amounts of accumulated DNA damage, measured as cyclobutane pyrimidine dimers (CPD) photoproducts, when exposed to UVR in the laboratory. Biological weighting functions (BWFs) revealed that S. neumayeri has significantly higher sensitivity to UVR-induced DNA damage across most of the UVR spectrum compared to the other two species, and all species were observed to have weightings in the ultraviolet-A (UVA, 320-400 nm) portion of the spectrum. The increased sensitivity to ultraviolet-B (290-320 nm) and UVA in S. neumayeri is correlated with the lowest concentration of UVR absorbing compounds observed in the embryos of the three species of urchin used in this study. Sea urchin embryos and larvae in the respective habitats of the species tested are known to occur within 5 m of the surface of the ocean where both UVB and UVA wavelengths occur. Solar irradiances of UVR at a depth of 5 m, weighted using the urchin DNA damage BWFs, show that E. chloroticus receives the greatest amount of biologically effective UVR despite having the lowest wavelength dependent weightings for DNA damage when compared to the other two species.     

Exposure to ultraviolet radiation (290-400 nm) causes oxidative stress, DNA damage, and expression of p53/p73 in laboratory experiments on embryos of the spotted salamander, Ambystoma maculatum

Developing embryos of the spotted salamander, Ambystoma maculatum, exposed to ultraviolet radiation (UVR; 290-400 nm) in the laboratory show a significant sensitivity to UVB (290-320 nm) radiation. Embryos in laboratory experiments exhibited significant DNA damage during exposures to UVR despite a significant increase in the production of the protective pigment melanin in response to UVR exposure. DNA damage occurs as a result of both the direct effects of exposure to UVR, and the indirect effects are mediated by the production of reduced oxygen intermediates. The production of reactive oxygen species initiates the expression of p53/p73 that leads to either DNA repair or apoptosis. When similar experiments are conducted on salamander embryos exposed to solar UVR in vernal pools, the embryos show significantly less sensitivity and higher survivorship. The differences between laboratory and field experiments are a result of the attenuation of UVR caused by the accumulation of dissolved organic carbon within the pools of these wooded areas. These findings suggest that northeastern populations of spotted salamanders are sensitive to UVR but are not significantly affected by present-day irradiances of UVR in the field. These results do suggest that continued decreases in stratospheric ozone over temperate latitudes have the potential to affect spotted salamanders in their natural habitats.     

Exposure to ultraviolet radiation causes proteomic changes in embryos of the purple sea urchin, Strongylocentrotus purpuratus

We performed experiments to determine how environmentally relevant ultraviolet radiation (UVR) affects protein expression during early development in the sea urchin, Strongylocentrotus purpuratus. To model the protein-mediated cell cycle response to UV-irradiation, six batches of embryos were exposed to UVR, monitored for both delays in the first mitotic division and changes in the proteome at two specific developmental time points. Embryos were exposed to or protected from artificial UVR (11.5 W/m²) for 25 or 60 min. These levels of UVR are within the range we have measured in coastal waters between 0.5 and 2 m. Embryos treated with UVR for 60 min cleaved an average of 23.2 min (± 1.92 s.e.m.) after UV-protected embryos. Differential protein spot migration between UV-protected and UV-treated embryos was examined at 30 and 90 min post-fertilization using two-dimensional SDS-PAGE (2D GE). A total of 1306 protein spots were detected in all gels, including differences in 171 protein spots (13% of the detected proteome) in UV-treated embryos at 30 min post-fertilization and 187 spots (14%) at 90 min post-fertilization (2-way ANOVA, P = 0.03, n = 6). The majority of the proteins affected by UVR were subsequently identified using matrix assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS). Our results indicate UVR affects proteins from multiple cellular pathways and indicate that the mechanisms involved in UV-stress and UV-induced developmental delay in sea urchin embryos are integrated among multiple pathways for cellular stress, protein turnover and translation, signal transduction, cytoskeletal dynamics, and general metabolism.     

The effects of some selected bacteria on embryos and larvae of the American oyster,Crassostrea virginica

An investigation was conducted to determine the effects of selected bacteria on healthy embryos and larvae of the American oyster, Crassostrea virginica. A total of 156 bacterial types were isolated from moribund larvae and particulate matter collected with a 36 μm Nitex screen. Most of the isolates were nonpigmented motile, gram-negative rods sensitive to chloramphenicol and neomycin, and were classified as Pseudomonas and Vibrio.Three oyster developmental stages were exposed to selected bacteria: embryos in the first 48 hr of development, 2-day-old larvae, and presetting, 10-day-old larvae. Twenty of the bacterial strains tested caused gross morphological abnormalities, decreased growth, and/or increased mortality for one or more of the three developmental stages. Neither culture filtrate nor heat-killed bacteria produced these effects.     

The effect of the quality of food patches on larval vertical distribution of the sea urchins Lytechinus variegatus (Lamarck) and Strongylocentrotus droebachiensis (Mueller)

Although most invertebrate larvae are weak swimmers and act as passive particles on horizontal scales, they may be able to regulate their vertical position in response to different factors, including increased food concentration. We examined the effect of the quality of food patches on larval vertical distribution for the sea urchins Lytechinus variegatus and Strongylocentrotus droebachiensis, and determined the effect of dietary conditioning on that response in the laboratory. We reared larvae on a mixed algal diet of Dunaliella tertiolecta and Isochrysis galbana under low (500 cells ml⁻¹) and high (5000 cells ml⁻¹) rations. Food patches were maintained in Plexiglas rectangular columns (30×10×10 cm) using a density gradient, where practical salinity in the bottom layer was 33, in the middle layer 30, and in the top layer 27. We examined the magnitude and mechanism of a behavioural response of larvae of L. variegatus in the four-arm stage, and on two developmental stages of S. droebachiensis (four- and six-arm), by manipulating patch quality. In the absence of a patch, larvae of both species and developmental stages swam through to the surface of the experimental columns. In the presence of algae, fewer larvae were present above the patch and more were at the patch than in control columns. More larvae swam through patches of “unflavoured” algal mimics than algal patches, and aggregated at the surface. Larval distribution relative to patches of algal filtrate without algal cells or of “flavoured” algal mimics in algal filtrate was not consistently different from that in either control or algal patches; thus, the magnitude of larval response to filtrate (with or without particles) was intermediate between that to control and algal patches. For L. variegatus, more larvae crossed the patches when reared on low than high rations, indicating that poorly conditioned larvae may be less responsive to environmental cues. Our results suggest that larvae can actively aggregate and maintain a vertical position in response to a food patch that depends on the quality and quantity of food. The response appears to be based mostly on a chemosensory rather than a mechanosensory mechanism.     

On barriers to hybridization between Strongylocentrotus droebachiensis (O.F. Müller) and S. Pallidus (G.O. Sars)

Strongylocentrotus droebachiensis (O. F. Müller) and Strongylocentrotus pallidus (G. O. Sars) occur together over much of their ranges in both the Atlantic and Pacific Oceans and remain distinct species, but no strong barrier to genetic exchange has been found in a study in the San Juan Islands. Co-occurrence of embryos of both species in the plankton indicates similar times of spawning. Ova of S. droebachiensis are fertilized by sperm of S. pallidus but the reciprocal cross gives a percent fertilization near zero. Hybrids of both crosses are viable with pigmentation resembling S. pallidus in the larval stage and S. droebachiensis in the adult stage. Ova of first generation female hybrids are more readily fertilized by sperm of S. pallidus, so that gene flow through female hybrids is more likely to be from S. droebachiensis to S. pallidus than the reverse. Male hybrids were not tested. S. droebachiensis and S. pallidus from deeper waters have lower fecundity than shallow water S. droebachiensis, which suggests that in the deeper waters individuals are food limited. The hypothesis that S. pallidus uses the sterile male technique to reduce reproductive output of S. droebachiensis is considered and rejected.     

Interactive effects of haloclines and food patches on the vertical distribution of 3 species of temperate invertebrate larvae

In laboratory experiments, we examined the effect of haloclines and determined whether the presence of food patches overrides this effect on larval vertical distribution of the sea star Asterias rubens, the sea urchin Strongylocentrotus droebachiensis and the mussel Mytilus edulis. We experimentally constructed haloclines in which the salinity of the bottom water layer was 35 and that of the top layer was 21, 24, 27, and 30 (21/35, 24/35, 27/35, and 30/35) for A. rubens and S. droebachiensis, and 24, 27, 30 and 32 (24/35, 27/35, 30/35, and 32/35) for M. edulis. For each species and stage, additional halocline treatments (A. rubens: 24/32 and 27/32; 4-arm S. droebachiensis: 21/29 and 24/32; 6-arm S. droebachiensis: 24/29 and 24/32; M. edulis: 27/32 and 30/32) were used to determine whether the larval response to inhibitory salinity gradients was due to the absolute salinity of the top layer or the relative salinity difference between the two layers. Also, we measured the density of A. rubens and M. edulis to determine whether the specific gravity of larvae can explain the observed vertical distributions. Larvae aggregated at and below the halocline and these aggregations were more pronounced with increasing strength of the vertical salinity gradient. Threshold salinities in the top layer which inhibited ~ 100% of the larvae from crossing the halocline were 24 for A. rubens and M. edulis, and 21 for S. droebachiensis. These distributional patterns were not the result of larval density, which was greater than all treatment water densities for M. edulis and S. droebachiensis and lower for A. rubens. The effect of the presence of a food patch at inhibitory haloclines (A. rubens: 24/35 and 27/35; 4-arm S. droebachiensis: 21/34 and 24/34; M. edulis: 27/35) was determined by using three algal densities: 0, 5000 or 10 000 cells ml^- 1Thalassiosira pseudonana in either the top or the bottom water layer. For both A. rubens and M. edulis, the number of larvae at the halocline increased in the presence of a food patch, but this effect did not depend on algal density in the patch. For 4-arm S. droebachiensis, there was no effect of the presence of a food patch on larval vertical distribution. Our results suggest that low salinity may act as a barrier to vertical movement and that the presence of food patches above the halocline may strengthen the larval aggregation response to inhibitory haloclines.     

Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint

Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of ph^p mutants reveals an acceleration of S phase, showing that ph^p regulates S phase length. Like ph^p mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of ph^p are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair.     

Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos

In most cells, the DNA damage checkpoint delays cell division when replication is stalled by DNA damage. In early Caenorhabditis elegans embryos, however, the checkpoint responds to developmental signals that control the timing of cell division, and checkpoint activation by nondevelopmental inputs disrupts cell cycle timing and causes embryonic lethality. Given this sensitivity to inappropriate checkpoint activation, we were interested in how embryos respond to DNA damage. We demonstrate that the checkpoint response to DNA damage is actively silenced in embryos but not in the germ line. Silencing requires rad-2, gei-17, and the polh-1 translesion DNA polymerase, which suppress replication fork stalling and thereby eliminate the checkpoint-activating signal. These results explain how checkpoint activation is restricted to developmental signals during embryogenesis and insulated from DNA damage. They also show that checkpoint activation is not an obligatory response to DNA damage and that pathways exist to bypass the checkpoint when survival depends on uninterrupted progression through the cell cycle.     

Tissue Damage Disrupts Developmental Progression and Ecdysteroid Biosynthesis in Drosophila

In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone – a steroid hormone that regulates developmental transitions in insects. We use Drosophila melanogaster as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition – a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.     

DNA preparation method in eggs, immature stages, and diapausing females of Tetranychus spider mites (Acari: Tetranychidae) for diagnostic PCR-RFLP

A DNA preparation method for diapausing females, immature stages, and eggs of spider mites for diagnostic analyses using restriction fragment length polymorphisms after polymerase chain reaction (PCR-RFLP analysis) of the internal transcribed spacer (ITS) region including 5.8 S of the nucleic ribosomal DNA was developed using Tetranychus urticae Koch as a typical example of tetranychid mites. In diapausing females and deutonymphs, the method of crushing an individual mite with a glass rod was eminently successful and a useful method to obtain the DNA template for PCR. For protonymphs, larvae, and eggs older than 48?h after oviposition at 24?°C, the DNA preparation method for a single nematode, crushing a tiny sample with a filter paper chip, was also successful and a useful method to obtain the DNA template from mite individuals (eggs) for PCR. Consequently, we found a single distinctive band of an amplified DNA fragment after electrophoresis in all developmental stages, and digestion of the PCR product by a restriction endonuclease, DraI, resulted in identical banding patterns being exhibited in all developmental stages. Our findings indicate that PCR-RFLP analysis of the ITS region can be used for species identification of diapausing females, immature stages, and eggs of Tetranychus species.     

DEVELOPMENTAL CONSEQUENCES OF AN EVOLUTIONARY CHANGE IN EGG SIZE: AN EXPERIMENTAL TEST

Larvae of two species of sea urchins (Strongylocentrotus droebachiensis and S. purpuratus) differ in initial form and in the rate of development. To determine whether these differences are attributable to the large interspecific difference in egg size, we experimentally reduced egg size by isolating blastomeres from embryos. The rate of development of feeding larvae derived from isolated blastomeres was quantified using a novel morphometric method. If the differences early in the life histories of these two species are due strictly to differences in egg size, then experimental reduction of the size of S. droebachiensis eggs should yield an initial larval form and rate of development similar to that of S. purpuratus. Our experimental manipulations of egg size produced three clear results: 1) smaller eggs yielded larvae that were smaller and had simpler body forms, 2) smaller eggs resulted in slower development through the early feeding larval stages, and 3) effects of egg size were restricted to early larval stages. Larvae from experimentally reduced eggs of the larger species had rates of development similar to those of the smaller species. Thus, cytoplasmic volumes of the eggs, not genetic differences expressed during development, account for differences in larval form and the rate of form change. This is the first definitive demonstration of the causal relationship between egg size (parental investment per offspring) and life-history characteristics in marine benthic invertebrates. Because larval form influences feeding capability, the epigenetic effects of egg size on larval form are likely to have important functional consequences. Adaptive evolution of egg size may be constrained by the developmental relationships between egg size and larval form: evolutionary changes in egg size alone can result in concerted changes in larval form and function; likewise evolutionary changes in larval form and function can be achieved through changes in egg size. These findings may have broader implications for other taxa in which larval morphology and, consequently, performance may be influenced by changes in egg size.     

Parasitism of Western Corn Rootworm Larvae and Pupae by Steinernema carpocapsae

Virulence and development of the insect-parasitic nematode, Steinernema carpocapsae (Weiser) (Mexican strain), were evaluated for the immature stages of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Third instar rootworm larvae were five times more susceptible to nematode infection than second instar larvae and 75 times more susceptible than first instar larvae and pupae, based on laboratory bioassays. Rootworm eggs were not susceptible. Nematode development was observed in all susceptible rootworm stages, but a complete life cycle was observed only in second and third instar larvae and pupae. Nematode size was affected by rootworm stage; the smallest infective-stage nematodes were recovered from second instar rootworm larvae. Results of this study suggest that S. carpocapsae should be applied when second and third instar rootworm larvae are predominant in the field.     

Susceptibility of Hoplia philanthus (Coleoptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida: Steinernematidae,Heterorhabditidae)

Biological control potential of nine entomopathogenic nematodes, Heterorhabditis bacteriophora CLO51 strain (HbCLO51), H. megidis VBM30 strain (HmVBM30), H. indica, Steinernema scarabaei, S. feltiae, S. arenarium, S. carpocapsae Belgian strain (ScBE), S. glaseri Belgian strain (SgBE) and S. glaseri NC strain (SgNC), was tested against second-, and third-instar larvae and pupae of Hoplia philanthus in laboratory and greenhouse experiments. The susceptibility of the developmental stages of H. philanthus differed greatly among tested nematode species/strains. In the laboratory experiments, SgBE, SgNC, HbCLO51 and HmVBM30 were highly virulent to third-instar larvae and pupae while SgBE was only virulent to second-instar larvae. Pupae were highly susceptible to HbCLO51, HmVBM30, SgBE and SgNC (57–100%) followed by H. indica and S. scarabaei (57–76%). In pot experiments, HbCLO51, SgBE and S. scarabaei were highly virulent to the third-instar larvae compared to the second-instar larvae. Our observations, combined with those of previous studies on other nematode and white grub species, show that nematode virulence against white grub developmental stages varies with white grub and nematode species.     

Diversity of Polyhydroxynaphthoquinone Pigments in North Pacific Sea Urchins

Using high‐performance liquid chromatography with diode‐array detection and mass spectrometry (HPLC‐DAD/MS) we investigated the composition of polyhydroxynaphthoquinone (PHNQ) pigments from sea urchins Strongylocentrotus pallidus, St. polyacanthus, St. droebachiensis, Brisaster latifrons and Echinarachnius parma, collected in the Sea of Okhotsk and the Bering Sea. Identification of PHNQ pigments from sea urchins St. polyacanthus, B. latifrons, and E. parma was performed for the first time. Among the usual PHNQ pigments, mono‐ and dimethoxy derivatives of spinochrome E, not previously found in other sea urchins, were discovered in St. polyacanthus and St. droebachiensis. In St. droebachiensis, two monomethoxy derivatives of echinochrome A were detected, isolated previously from only tropical sea urchins. It was found that the composition and total content of pigments of St. droebachiensis depends on the collection area of the sea urchins and its depth and varies from 88 to 331 μg/g of dry shells. Sea urchins St. pallidus, B. latifrons and E. parma had average values for PHNQ pigment content, approximately 30 μg/g, and St. polyacanthus had a low PHNQ content, 13 μg/g.     

The effect of prey density on the developmental time of larvae of an aquatic beetle: <Emphasis Type="Italic">Tropisternus setiger</Emphasis> (Insecta,Coleoptera: Hydrophilidae)

Predaceous larvae of the water scavenger beetle Tropisternus setiger (Germar) are common inhabitants of variable environments in which prey availability may vary widely. We conducted laboratory experiments to assess the effect of prey density on developmental times and survivorship of the preimaginal stages of T. setiger. We also examined the effect of the number of consumed prey on the larval size of instar III. Four different prey densities (one, two, four, and eight preys a day) were tested and both developmental time and survivorship differed significantly among them. Larvae fed one or two preys daily showed a longer developmental time and a lower survivorship than larvae fed four or eight preys a day. Moreover the consumption of four preys a day increased larval developmental success, and to consume one prey a day affected survivorship through the larval period. On the other hand, prey density had no effect on the final larval size. Handling editor: K. Martens