Use of Antibacterial Agents To Elucidate the Etiology of Juvenile Oyster Disease (JOD) in Crassostrea virginica and Numerical Dominance of an α-Proteobacterium in JOD-Affected Animals

Since 1988, juvenile oyster disease (JOD) has resulted in high seasonal losses of cultured Eastern oysters (Crassostrea virginica) in the Northeast. Although the cause of JOD remains unknown, most evidence is consistent with either a bacterial or a protistan etiology. For the purpose of discerning between these hypotheses, the antibacterial antibiotics norfloxacin and sulfadimethoxine-ormetoprim (Romet-B) were tested for the ability to delay the onset of JOD mortality and/or reduce the JOD mortality of cultured juvenile C. virginica. Hatchery-produced C. virginica seed were exposed in triplicate groups of 3,000 animals each to either norfloxacin, sulfadimethoxine-ormetoprim, or filter-sterilized seawater (FSSW) and deployed in floating trays on the Damariscotta River of Maine on 17 July 1997. Each week thereafter, a subset of animals from each group was reexposed to the assigned treatment. Repeated immersion in either a sulfadimethoxine-ormetoprim or a norfloxacin solution resulted in a delay in the onset of JOD mortality in treated animals and reduced weekly mortality rates. Weekly treatments with either norfloxacin or sulfadimethoxine-ormetoprim also resulted in a statistically significant reduction in cumulative mortality (55 and 67% respectively) compared to animals treated weekly with FSSW (81%) or those that had received only a single treatment with either norfloxacin, sulfadimethoxine-ormetoprim, or FSSW (77, 84, and 82%, respectively). Bacteriological analyses revealed a numerically dominant bacterium in those animals with obvious signs of JOD. Sequence analysis of the 16S rRNA gene from these bacteria indicates that they are a previously undescribed species of marine α-proteobacteria.     

Epizootiology and pathology of juvenile oyster disease in the Eastern oyster, Crassostrea virginica.

Juvenile Oyster Disease (JOD) causes mortalities of small cultured oysters, Crassostrea virginica. The present study was an intensive epizootiological and pathological investigation of JOD in eight sequentially deployed cohorts at sites on Long Island, New York. JOD symptoms and mortalities began in all groups at about the same time. Lesions on the mantle were detected histologically about 1 week before the principal symptom, a conchiolin deposit on the inner shell, appeared. Mortality began about 1 week later and reached 60-90% in oysters <25 mm. Mantle lesions were highly correlated with subsequent conchiolin-deposit prevalence and with total mortality. Larger juveniles (25-40 mm) were affected by the disease and produced conchiolin deposits, but mortalities did not exceed 30%. Mortalities were consistently related to size, but not necessarily to age or length of "exposure" in the field. There was no indication that JOD was linked to a particular broodstock or hatchery. Wild spat deployed at experimental sites showed JOD symptoms before the hatchery-produced groups did and cohorts maintained inside a hatchery experienced essentially no JOD. Histological examination of cohorts experiencing high mortalities failed to reveal an obvious etiological agent, but showed a disease pattern similar to that described for other bivalve diseases with a bacterial etiology. Similarities and differences between this and other studies of JOD suggest that one or more bacterial species is responsible for JOD, but that a trigger, probably temperature, is also involved and may vary from site to site.     

Interactions of ecdysteroid and juvenoid agonists in Plodia interpunctella (Hübner)

The influence of non-steroidal ecdysteroid agonists on Indianmeal moth larvae was assessed by rearing last instar larvae on diet treated with RH-5992 (tebufenozide) or RH-2485 (methoxyfenozide). Larvae were monitored for effects of the ecdysteroid agonists on weight, metamorphosis and mortality. Larvae treated with either of the ecdysteroid agonists at a concentration of 5 ppm or higher gained less weight and had greater mortality than did larvae reared on control diet. For example, the weights of control larvae increased approximately 400% by day 2, compared with only a 50% increase in weight when the larvae were treated with 25 ppm of RH-2485 or RH-5992. Similarly, mortality in control larvae was less than 10%, but was as much as 90–100% in larvae reared on diet treated with one of the ecdysteroid agonists. We also examined the effects of simultaneous treatment with a juvenile hormone (JH) mimic, either methoprene or fenoxycarb. The JH mimics prevented adult emergence, and the larvae continued to feed throughout the month-long observation period. However, larvae treated with a juvenile hormone mimic gained weight despite the presence of an ecdysteroid agonist in the diet. On diets treated with 0.1 ppm of RH-2485 or RH-5992, JH-treated larvae gained even more weight than did untreated controls. Interestingly, although the addition of a JH mimic to ecdysteroid-treated diet resulted in increased weight, it did not lead to reduced mortality. In fact, combinations of a JH mimic with 10 ppm RH 2485 or RH 5992 resulted in nearly 100% mortality compared with 40–70% mortality without the JH compounds. These results indicate that JH mimics overcome the inhibitory effects of ecdysteroid agonists on weight gain; however, they also resulted in increased mortality compared with moderate doses of ecdysteroid agonists alone. One specific action of these compounds at the cellular level was noted in that RH 5992 mimicked ecdysteroids by increasing uptake of ¹⁴C-GlcNAc in a Plodia interpunctella cell line, while fenoxycarb was inhibitory. Arch. Insect Biochem. Physiol. 38:91–99, 1998. Published 1998 Wiley-Liss, Inc.     

Use of the 16S-23S rDNA internal transcribed spacer of Roseovarius crassostreae for epizootiological studies of juvenile oyster disease (JOD)

Juvenile oyster disease (JOD) in Crassostrea virginica is caused by the marine bacterium Roseovarius crassostreae. Although the 16S rRNA genes of the bacterial isolates exhibit little variation, 2 genetic signatures (GSI and GSII) may be discerned by Ava I digestion of the 16S-23S internal transcribed spacer (ITS). In this study we analyzed isolates from JOD epizootics throughout the northeastern USA (including affected adults for the first time) to better understand how oyster populations encounter and become affected by the pathogen. Isolates from a given epizootic usually had the same ITS signature; however, the involvement of both genetic signatures was occasionally detected, even within the same oyster. Sequencing was used to localize the variable Ava I site to a 100 bp region of low sequence identity, and detection of additional base changes resulted in the identification of 11 distinct genotypes. One genotype was found only in Martha's Vineyard, Massachusetts, USA and persisted in JOD survivors. Two genotypes were associated with Maine epizootics, and both were believed to be unique to that region until 2004, when one was detected in Martha's Vineyard among oysters that had survived colonization by the local genotype. Apparent competition between those 2 genotypes was also detected among a population of juveniles. Five genotypes were found only in New York, and the other 3 were isolated from both New York and from around Cape Cod, Massachusetts. Relationships between the geographic occurrence and phylogenetic relatedness of genotypes were compared with regional current patterns to identify possible mechanisms controlling their distribution.     

Transmission of the haplosporidian parasite MSX Haplosporidium nelsoni to the eastern oyster Crassostrea virginica in an upweller system

The haplosporidian oyster parasite MSX (Multinucleated Sphere X) Haplosporidium nelsoni was transmitted to eastern oysters Crassostrea virginica. Hatchery-raised, MSX-free juvenile oysters were placed in upweller tanks. Water to the tanks was filtered through a screen with 1 mm2 openings and originated from the water column overlaying naturally infected oysters beds (MSX prevalence 17 to 57%). MSX was diagnosed by histopathological analysis. MSX-disease (57% prevalence) with increased mortality (19%) was observed 11 wk after the beginning of the exposure and mortality of 80% after 16 wk. The study demonstrates transmission of MSX via water-borne infectious agents capable of passing through a 1 mm filter.     

A PCR-based diagnostic assay for the detection of Roseovarius crassostreae in Crassostrea virginica affected by juvenile oyster disease (JOD)

We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD.     

Competition in size-structured populations: mechanisms inducing cohort formation and population cycles

In this paper we investigate the consequences of size-dependent competition among the individuals of a consumer population by analyzing the dynamic properties of a physiologically structured population model. Only 2 size-classes of individuals are distinguished: juveniles and adults. Juveniles and adults both feed on one and the same resource and hence interact by means of exploitative competition. Juvenile individuals allocate all assimilated energy into development and mature on reaching a fixed developmental threshold. The combination of this fixed threshold and the resource-dependent developmental rate, implies that the juvenile delay between birth and the onset of reproduction may vary in time. Adult individuals allocate all assimilated energy to reproduction. Mortality of both juveniles and adults is assumed to be inversely proportional to the amount of energy assimilated. In this setting we study how the dynamics of the population are influenced by the relative foraging capabilities of juveniles and adults.In line with results that we previously obtained in size-structured consumer-resource models with pulsed reproduction, population cycles primarily occur when either juveniles or adults have a distinct competitive advantage. When adults have a larger per capita feeding rate and are hence competitively superior to juveniles, population oscillations occur that are primarily induced by the fact that the duration of the juvenile period changes with changing food conditions. These cycles do not occur when the juvenile delay is a fixed parameter. When juveniles are competitively superior, two different types of population fluctuations can occur: (1) rapid, low-amplitude fluctuations having a period of half the juvenile delay and (2) slow, large-amplitude fluctuations characterized by a period, which is roughly equal to the juvenile delay. The analysis of simplified versions of the structured model indicates that these two types of oscillations also occur if mortality and/or development is independent of food density, i.e. in a situation with a constant juvenile developmental delay and a constant, food-independent background mortality. Thus, the oscillations that occur when juveniles are more competitive are induced by the juvenile delay per se. When juveniles exert a larger foraging pressure on the shared resource, maturation implies an increase not only in adult density, but also in food density and consequently fecundity. Our analysis suggests that this correlation in time between adult density and fecundity is crucial for the occurrence of population cycles when juveniles are competitively superior.     

Localization of the bacterial agent of juvenile oyster disease (Roseovarius crassostreae) within affected eastern oysters (Crassostrea virginica)

The bacterium Roseovarius crassostreae causes seasonal mortalities among commercially produced eastern oysters (Crassostrea virginica) grown in the Northeastern United States. Phylogenetically, the species belongs to a major lineage of marine bacteria (the Roseobacter clade), within which Roseovarius crassostreae is the only known pathogen to be isolated in laboratory culture. The objective of the current study was to determine the location and nature of R. crassostreae interactions with oysters affected by juvenile oyster disease (JOD). Scanning electron microscopy of diseased individuals revealed abundant colonization of the inner shell surfaces by bacteria which were morphologically similar to R. crassostreae. The same types of cells were also observed on and within layers of host-derived conchiolin on the inner valves. Most bacterial cells were alive as determined by the use of a fluorescent viability stain. Further, most were clearly attached at the cell poles, which is consistent with the ability of R. crassostreae to express polar fimbriae. When material from the pallial fluid, soft tissue and inner valve surfaces was cultured, the highest numbers of R. crassostreae were recovered from the inner valves. These samples also contained the greatest abundance of R. crassostreae as a percentage of total colonies. Cloning and sequencing of 16S rRNA genes provided culture-independent evidence of the numerical dominance of R. crassostreae among the bacterial consortia associated with the inner shell surfaces of JOD-affected animals. The ability of R. crassostreae to colonize shell and conchiolin is consistent with the described JOD-pathology and may aid the bacteria in avoiding hemocyte-mediated killing.     

Severe Hypoglycemia in a Juvenile Diabetic Rat Model: Presence and Severity of Seizures Are Associated with Mortality

It is well accepted that insulin-induced hypoglycemia can result in seizures. However, the effects of the seizures, as well as possible treatment strategies, have yet to be elucidated, particularly in juvenile or insulin-dependent diabetes mellitus (IDDM). Here we establish a model of diabetes in young rats, to examine the consequences of severe hypoglycemia in this age group; particularly seizures and mortality. Diabetes was induced in post-weaned 22-day-old Sprague-Dawley rats by streptozotocin (STZ) administered intraperitoneally (IP). Insulin IP (15 U/kg), in rats fasted (14–16 hours), induced hypoglycemia, defined as <3.5 mM blood glucose (BG), in 68% of diabetic (STZ) and 86% of control rats (CON). Seizures occurred in 86% of STZ and all CON rats that reached hypoglycemic levels with mortality only occurring post-seizure. The fasting BG levels were significantly higher in STZ (12.4±1.3 mM) than in CON rodents (6.3±0.3 mM), resulting in earlier onset of hypoglycemia and seizures in the CON group. However, the BG at seizure onset was statistically similar between STZ (1.8±0.2 mM) and CON animals (1.6±0.1 mM) as well as between those that survived (S+S) and those that died (S+M) post-seizure. Despite this, the S+M group underwent a significantly greater number of seizure events than the S+S group. 25% glucose administered at seizure onset and repeated with recurrent seizures was not sufficient to mitigate these continued convulsions. Combining glucose with diazepam and phenytoin significantly decreased post-treatment seizures, but not mortality. Intracranial electroencephalograms (EEGs) were recorded in 10 CON and 9 STZ animals. Predictive EEG changes were not observed in these animals that underwent seizures. Fluorojade staining revealed damaged cells in non-seizing STZ animals and in STZ and CON animals post-seizure. In summary, this model of hypoglycemia and seizures in juvenile diabetic rats provides a paradigm for further study of underlying mechanisms. Our data demonstrate that severe hypoglycemia (<2.0 mM) is a necessary precondition for seizures, and the increased frequency of these seizures is associated with mortality.     

Impact of vineyard pesticides on a beneficial arthropod, Typhlodromus pyri (Acari: Phytoseiidae), in laboratory bioassays

Laboratory bioassays were conducted to evaluate the effects of six vineyard pesticides on Typhlodromus pyri Scheuten (Acari: Phytoseiidae), a key predator of the mite Calepitrimerus vitis Nalepa (Acari: Eriophyoidae), in Pacific coastal vineyards. Materials tested were whey powder, 25% boscalid + 13% pyraclostrobin (Pristine), 40% myclobutanil (Rally), micronized sulfur (92% WP), 75% ethylene bisdithiocarbamate (mancozeb; Manzate), and 91.2% paraffinic oil (JMS Stylet), all applied at three concentrations. Pesticide dilutions were directly sprayed onto T. pyri adult females and juveniles, and each treatment was assessed to determine effects on direct mortality and fecundity. Five of the six pesticides tested resulted in < 50% mortality to adult and juvenile T. pyri for all concentrations 7 d after treatment. Paraffinic oil treatments displayed direct mortality > 50% for adult and juvenile assays and resulted in significantly higher mortality. Sublethal effects were more pronounced than acute pesticide toxicity, particularly in juvenile mite bioassays. Significant decreases in fecundity were detected in the sulfur and mancozeb treatments compared with the control in juvenile tests. The relative percentage of fecundity reduction for juvenile mites was highest when applying mancozeb (> 70%), sulfur (> 25%), or myclobutanil (> 20%). Adult mites displayed the greatest reductions in fecundity from applications of paraffinic oil (> 20%) or mancozeb (> 15%) treatments. Boscalid (+ pyraclostrobin) and whey displayed the least effect on fecundity across all bioassays. These results can be used to develop management guidelines in vineyard pest management practices to help conserve and enhance predatory mite populations.     

Embryotoxicity studies of norfloxacin in cynomolgus monkeys. II. Role of progesterone.

Norfloxacin, an orally active fluoroquinolone antimicrobial, has been reported to be embryolethal but not teratogenic when administered to pregnant cynomolgus macaques prior to gestational day (GD) 36 at doses > or = 200 mg/kg/day. Additional studies have been performed in an effort to examine the mechanism responsible for this effect, particularly regarding the role of progesterone (P). The first study (Study I) investigated the effect of norfloxacin administration during early pregnancy (200 mg/kg/day; daily GD 20-30) in the absence of a functional corpus luteum (CL). The CL was surgically removed from 16 gravid females on GD 19 in order to focus on placental-derived P; ten were dosed with norfloxacin and six received vehicle only. Embryolethality was observed for 7/10 (70%) of the treated animals during GD 25-31 versus 0/6 (0%) for controls. A reduction in serum P was noted prior to embryonic loss, although no significant effects on chorionic gonadotropin (CG), 17 beta-estradiol (E2), or P or E urinary metabolites were observed. A second study (Study II) was performed in order to evaluate the capacity of norfloxacin (200 mg/kg) to reduce CL-derived P in both normally cycling and CG-stimulated nonpregnant females (ten treated, ten controls; daily for 8 days). No effects on P production or on luteal phase or menstrual cycle lengths were observed. The third study (Study III) was designed to examine the effect of norfloxacin on the metabolism and excretion of P in nonpregnant females. Silastic P implants were placed subcutaneously in order to maintain constant P levels during a 10 day treatment regimen (200 mg/kg/day; ten controls, nine treated). Five of the controls and four of the norfloxacin-treated females also received 14C-P intravenously within 1 hr of the last dose of norfloxacin in order to study excretory patterns. No significant differences between control and treated groups were observed. The results of these studies combined suggest that the developmental toxic effects observed in prior studies and Study I are specific to pregnancy and directly related to placental-derived P production.     

Geographic differences for delay of sexual maturation in Peromyscus leucopus: effects of photoperiod, pinealectomy, and melatonin

Effects of short-day photoperiod, pinealectomy, and melatonin on sexual maturation were tested in Peromyscus leucopus from either Connecticut (CT) or Georgia (GA). Laboratory reared-stocks from CT and GA were exposed to short daylength (photoperiod) from birth or 25 days of age. At 12 wk of age, delay in sexual maturation was indicated in most CT mice by decreased testis length, combined testes weight, and seminal vesicle weight. Conversely, GA animals did not delay sexual maturation when exposed to short-day photoperiod from either birth or 25 days of age. These results indicate that responses to short daylengths differ for juvenile CT and GA populations. In a second experiment, pinealectomized or sham-operated CT males were exposed to short-day (9L:15D) or long-day (16L:8D) photoperiod from birth. Pinealectomy blocked the effect of short daylength on reproduction. Therefore, the pineal must be involved in the delay of sexual maturation observed for short-day CT mice. The effects of melatonin, a pineal gland hormone, were tested with chronic s.c. implants or daily injections. In CT mice given either melatonin implants or afternoon injections, sexual maturation was delayed. GA mice were insensitive to all melatonin treatments. Further, no differences in circadian organization (phase angle, duration of activity, period under constant dark) between GA and CT animals were apparent. Collectively, these studies indicate that melatonin is involved in the mechanism responsible for delay of sexual maturation in CT mice. Short-day insensitivity of GA Peromyscus leucopus probably results from a deficiency in the melatonin effector pathway and is not due to a disruption of circadian organization.     

Circadian photoentrainment: parameters of phase delaying

Experiments were carried out using simulated den cages to delineate specific characteristics of phase delaying in circadian photoentrainment of a nocturnal rodent, the flying squirrel. The principal experiments entailed presentation of one to five consecutive 15-min white-light pulses per activity cycle at activity onset to animals free-running in darkness, in order to determine the immediate and final phase-shifting effect. Auxiliary experiments recorded entrainment patterns on light-dark (LD) schedules in the den cages. Phase response curves (PRCs) based on 15-min white-light pulses in standard wheel cages were also constructed for these animals as background information for interpreting the phase-delaying experiments. Exposure of a den animal to light by light sampling at the time of initial arousal from the rest state at circadian time (CT) 12, either by an LD schedule or by a 15-min light pulse, resulted in a return to the nest box for a short rest period. The phase delay occurring after a single light exposure at activity onset was equal to the induced rest, thus suggesting an immediate phase shift. The maximum delay was about 1 1/2 hr/cycle, with the amount of delay related to the number of light exposures. During the photoentrained state on an LD schedule, the activity rhythm of a den-housed animal was essentially free-running on the days following a phase delay. The data are used to expand current models for photoentrainment of circadian activity rhythms in nocturnal rodents.     

Renal tubular sites of increased phosphate transport and NaPi-2 expression in the juvenile rat

To determine the tubular sites and mechanisms involved in enhanced renal phosphate (P(i)) reabsorption seen in the juvenile animal, renal micropuncture experiments were performed in acutely thyroparathyroidectomized adult (>14 wk old) and juvenile (4 wk old) male Wistar rats fed either a normal P(i) diet (NPD, 0.6% P(i)) or low P(i) diet (0.07% P(i)) for 2 days, in the presence and absence of parathyroid hormone (PTH). P(i) reabsorption was greater in proximal convoluted (PCT) and straight tubules (PST) of the juvenile compared with adult rats fed NPD, whether or not PTH was present. These findings were consistent with a greater P(i) uptake in brush-border membrane (BBM) vesicles from both superficial (SC) and outer juxtamedullary (JMC) cortices of juvenile animals. Western blot analysis revealed a 2- and 1.8-fold higher amount of NaPi-2 protein in the SC and JMC, respectively, in juvenile rats. Immunofluorescence microscopy also indicated that NaPi-2 protein expression was present in the proximal tubule (PT) BBM to a greater extent in juvenile rats. Dietary P(i) restriction in juvenile rats resulted in a significant increase in P(i) reabsorption in the PCT and PST segments. NaPi-2 expression in the PT BBM was also increased, as was the expression of intracellular NaPi-2 protein. These studies indicate that P(i) reabsorption in both the PCT and PST segments of the renal tubule contributes to the attenuated response to PTH in the normal juvenile animal. In addition, dietary P(i) restriction in the juvenile rat upregulates BBM NaPi-2 expression, which is associated with a further increase in proximal tubular P(i) reabsorption.     

Absorbable deslorelin implants (Ovuplant) prolong postpartum anestrus in early ovulating dairy cows

Two experiments were conducted to investigate the use of a bioabsorbable implant of the GnRH agonist deslorelin to temporarily delay the resumption of postpartum ovulatory cycles in Holstein cows. In Experiment 1, recently calved cows were paired and received either a single implant (Ovuplant); Peptech Animal Health, Sydney, NSW, Australia) within 48 h of parturition (OVP; n=17), or remained as untreated controls (CON; n=17). Blood samples were collected for plasma progesterone assay three times weekly for 6 weeks to profile the pattern of resumption of ovulatory cycles. In Experiment 2, there were 15 CON and 15 OVP cows initially treated as for Experiment 1 as well as 15 OVP+SYNCH cows. Each cow in the CON and OVP+SYNCH groups received a progesterone vaginal insert (CIDR); Genetics Australia, Bacchus Marsh, Vic., Australia) for 7 days at 23 days postpartum (23 dpp) to synchronise estrus in cycling animals or to induce an ovulation with estrus in anestrus animals. Blood samples were collected weekly until removal of the CIDR insert, and then twice weekly until 56 dpp to monitor plasma P4 for retrospective determination of ovulation. Milk yield was monitored by twice daily electronic volume measurements and milk composition with once weekly milk composition analysis.In Experiment 1, CON cows began ovulating from 9 dpp; 15 of 17 had ovulated by the end of blood sampling at 42 dpp. None of the OVP cows ovulated until at least 24 dpp, and only 6 of 17 had ovulated by 42 dpp. The average day of first ovulation was extended from 22.4+/-2.7 dpp to 39.3+/-2.7 dpp (P<0.05). In Experiment 2, ovulation had occurred in 8 of 15 CON cows at the time of CIDR insertion (23 dpp), 0 of 15 OVP cows and 1 of 15 OVP+SYNCH cows. By 40 dpp (or 10 days following removal of the CIDR insert) every CON cow (15/15) had ovulated, but only 2 of 15 OVP+SYNCH cows and 1 of 15 OVP cows. None of these effects of treatment was associated with any changes in milk yield or composition in either experiment.In conclusion, inserting a bioabsorbable implant of deslorelin within 48 postpartum extended the interval to first ovulation to at least 24 dpp in 46 of 47 cows. Recovery periods were highly variable. This variability was not reduced by using a form of intravaginal progesterone supplementation that did produce a synchronised estrus with ovulation in anestrus animals that had not been treated with deslorelin.     

Unexpected discontinuities in life-history evolution under size-dependent mortality

In many organisms survival depends on body size. We investigate the implications of size-selective mortality on life-history evolution by introducing and analysing a new and particularly flexible life-history model with the following key features: the lengths of growth and reproductive periods in successive reproductive cycles can vary evolutionarily, the model does not constrain evolution to patterns of either determinate or indeterminate growth, and lifetime number and sizes of broods are the outcomes of evolutionarily optimal life-history decisions. We find that small changes in environmental conditions can lead to abrupt transitions in optimal life histories when size-dependent mortality is sufficiently strong. Such discontinuous switching results from antagonistic selection pressures and occurs between strategies of early maturation with short reproductive periods and late maturation with long reproductive cycles. When mortality is size-selective and the size-independent component is not too high, selection favours prolonged juvenile growth, thereby allowing individuals to reach a mortality refuge at large body size before the onset of reproduction. When either component of mortality is then increased, the mortality refuge first becomes unattractive and eventually closes up altogether, resulting in short juvenile growth and frequent reproduction. Our results suggest a new mechanism for the evolution of life-history dimorphisms.     

The costs of phenotypic adaptation to repeatedly fluctuating temperatures in a soil arthropod

Costs of phenotypic adaptation to changing environments have often been studied in morphological structures. Such structures typically are irreversible for at least some stage in the organism's life. In this study we investigated whether recurrent and reversible adaptation to changes in the thermal environment incurs a cost in terms of some key life-history traits in the collembolan Orchesella cincta. We exposed juvenile O. cincta to two treatments differing in the frequency of temperature fluctuation but with equal total temperature sums. In the high frequency treatment temperature fluctuated daily between 10 and 20 °C, while in the low frequency treatment temperature fluctuated on a weekly schedule. During the treatments we measured juvenile growth rate and juvenile mortality, and after six weeks the animals were transferred to constant 15 °C and adult starvation resistance was assessed. We found no significant differences between the treatments in juvenile growth rate or juvenile survival. Also, adults that had grown up under high frequency temperature fluctuations did not suffer from reduced starvation resistance compared to animals growing under low frequency temperature fluctuations. This finding supports the hypothesis that selection minimizes the production costs of inducible phenotypes and suggests that the development of optimal phenotypes and evolution of temperature reaction norms are not constrained by such costs.     

Assessment of the northern distribution range of selected Perkinsus species in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) with the use of PCR-based detection assays

Perkinsus species are protistan parasites of molluscs. In Chesapeake Bay, Perkinsus marinus, Perkinsus chesapeaki, and Perkinsus andrewsi are sympatric, infecting oysters and clams. Although P. marinus is a pathogen for Crassostrea virginica, it remains unknown whether P. andrewsi and P. chesapeaki are equally pathogenic. Perkinsus species have been reported in C. virginica as far north as Maine, sometimes associated with high prevalence, but low mortality. Thus, we hypothesized that, in addition to P. marinus, Perkinsus species with little or no pathogenicity for C. virginica may be present. Accordingly, we investigated the distribution of Perkinsus species in C. virginica and Mercenaria mercenaria, collected from Maine to Virginia, by applying PCR-based assays specific for P. marinus, P. andrewsi, and a Perkinsus sp. isolated from M. mercenaria. DNA samples of M. mercenaria possessed potent PCR inhibitory activity, which was overcome by the addition of 1 mg/ml BSA and 5% (v/v) DMSO to the PCR reaction mixture. All 3 Perkinsus species were found in both host species throughout the study area. Interestingly, the prevalence of P. marinus in M. mercenaria was significantly lower than in C. virginica, suggesting that M. mercenaria is not an optimal host for P. marinus.     

Detection of the oyster herpesvirus in commercial bivalve in northern California, USA: conventional and quantitative PCR

The ostreid herpesvirus (OsHV-1) and related oyster herpesviruses (OsHV) are associated with world-wide mortalities of larval and juvenile bivalves. To quantify OsHV viral loads in mollusc tissues, we developed a SYBR Green quantitative PCR (qPCR) based on the A-region of the OsHV-1 genome. Reaction efficiency and precision were demonstrated using a plasmid standard curve. The analytical sensitivity is 1 copy per reaction. We collected Crassostrea gigas, C. sikamea, C. virginica, Ostrea edulis, O. lurida, Mytilus galloprovincialis, and Venerupis phillipinarum from Tomales Bay (TB), and C. gigas from Drakes Estero (DE), California, U.S.A., and initially used conventional PCR (cPCR) to test for presence of OsHV DNA. Subsequently, viral loads were quantified in selected samples of all tested bivalves except O. lurida. Copy numbers were low in each species tested but were significantly greater in C. gigas (p < 0.0001) compared to all other species, suggesting a higher level of infection. OsHV DNA was detected with cPCR and/or qPCR and confirmed by sequencing in C. gigas, C. sikamea, C. virginica, O. edulis, M. galloprovincialis, and V phillipinarum from TB and C. gigas from DE. These data indicate that multiple bivalve species may act as reservoirs for OsHV in TB. A lack of histological abnormalities in potential reservoirs requires alternative methods for their identification. Further investigation is needed to determine the host-parasite relationship for each potential reservoir, including characterization of viral loads and their relationship with infection (via in situ hybridization), assessments of mortality, and host responses.