Communal larval rearing of European lobster (Homarus gammarus): family identification by microsatellite DNA profiling an offspring fitness.

Stock enhancement experiments of European lobster (Homarus gammarus) have been carried out around the Kvitsoy Islands in south-western Norway since 1990. In addition to releases of coded wire tagged lobster juveniles (cultured) and subsequent monitoring of commercial fishery, a lobster hatchery was established in 1997. Several experiments were made on the communal-rearing approach where the performance of mixed larval groups (families) was evaluated under identical conditions. Berried females of wild and cultured origin and their respective fertilised eggs were screened by using microsatellite DNA profiling involving a multiplex set of six lobster specific primers, thereby allowing determination of both parental genotypes. Each female were kept separately during hatching, and the offspring were later mixed and raised in a communal rearing system. The early-larval survival was estimated at stage IV (bottom stage), and the survivors were identified to family and group by microsatellite profiling. Five different communal experiments were conducted, representing offspring from 65 berried females. Of the surviving larvae, 6.3% could not be assigned to family due to degraded DNA and no PCR amplification. Significant differences in early survival between offspring of wild and cultured origin were found in the experiments. No differences between the groups were found in stage IV larval size. Based on the pooled data on survival (as a measure of early larvae fitness) offspring of cultured females displayed a relative fitness of 60% in comparison to offspring from wild females. Large variation in survival was also observed among families within the wild and cultured groups, suggesting a genetic component for these traits and a potential for selective breeding.

Female reproductive biology of two sympatric incirrate octopod species, Adelieledone polymorpha (Robson 1930) and Pareledone turqueti (Joubin 1905) (Cephalopoda : Octopodidae), from South Georgia

The reproductive biology of two species of endemic Southern Ocean octopods was investigated around the sub-Antarctic islands of South Georgia and Shag Rocks. The females of both the species produce few, large eggs. This appears to be governed by phylogenetic constraint. No evidence was found for ontogenetic migration or seasonality associated with gonad maturation. Based on oocyte length frequency distributions and observations of oocyte development within the ovary, it is possible that both species could have either a single or multiple-batch spawning strategy. Pareledone turqueti ovaries contained fewer larger oocytes than those of Adefieledone polymorpha, which may help to reduce competition for resources immediately after hatching.

Chemosensory learning in the chicken embryo

Examined how exposure to a chemosensory stimulus prior to hatching affects subsequent chemosensory preferences of newly hatched chicks. The chicks' preferences were assessed at 2 days after hatching using an olfactory preference test (strawberry-smelling shavings vs water-coated shavings) and at 4 days after hatching using a gustatory preference test (strawberry-flavoured water vs unflavoured water). With no exposure to strawberry before hatching, strawberry was highly aversive to chicks after hatching. However, following exposure to strawberry before hatching, chicks expressed a greater preference for (or weaker aversion to) the strawberry stimulus. Chicks exposed to strawberry before hatching drank more strawberry-flavoured water and spent more time in a strawberry-scented area than chicks having no exposure before hatching. This change in preference was specific to the stimulus experienced before hatching and was present in the absence of any posthatching exposure to the stimulus. Results demonstrate that a chick's chemosensory preferences are changed as a result of experience with a stimulus before hatching and are suggestive of learning.

Avoidance of filial cannibalism in the amphipod Gammarus pulex.

Adult animals that cannibalise juvenile conspecifics may gain energy but also risk filial cannibalism, that is, consumption of their own offspring. However, individuals vary in the magnitude of the costs and benefits of cannibalism depending on factors such as their current energy reserves or the probability that they have offspring in the vicinity. They may therefore also vary in the extent to which they participate in cannibalism. This study investigated whether the sex or brooding status of adult amphipods (Gammarus pulex) influenced whether they participated in cannibalism of juveniles. For females carrying embryos within their brood pouch, we also investigated two hypotheses to explain the presence or absence of cannibalistic behaviour by determining whether cannibalism was correlated with factors that might reflect energy demands (body length, brood size), or that might reflect a temporal change in cannibalistic behaviour (corresponding to stage of brood development). All reproductive classes of adults participated in some level of juvenile cannibalism, but females carrying offspring at an advanced stage of development (close to emergence from the brood pouch) consumed significantly fewer juveniles than other groups. Females thus appear to significantly reduce cannibalism of juveniles concurrent with the time when their own eggs are hatching within the brood pouch, prior to the release of their offspring. Because the experiment tested female responses to unfamiliar juveniles, this reflects a temporal change in behaviour rather than a response to phenotypic recognition cues, although additional direct recognition cannot be ruled out. Brooding females with large brood sizes or large body lengths, which might have disproportionately greater energetic demands, were not more likely to cannibalise juveniles. We also noted that juveniles that survived in trials where cannibalism occurred were significantly more likely to be found at the water surface, suggesting a possible adaptation to escape cannibalistic adults. Overall, our results provide evidence that amphipods use indirect temporal cues to avoid filial cannibalism.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Thermal conditions in nests of loggerhead turtles: further evidence suggesting female skewed sex ratios of hatchling production in the Mediterranean

Temperature was recorded in 23 nests of the loggerhead turtle (Caretta caretta) and control sites of nest depth at Alagadi (35 degrees 33'N, 33 degrees 47'E), Northern Cyprus, eastern Mediterranean. Control site sand temperature was found to be highly correlated with mean daily air temperature and mean nest temperature. Mean temperature in nests ranged from 29.5 degreesC to 33.2 degreesC, with mean temperature in the middle third of incubation ranging from 29.3 degreesC to 33.7 degreesC. Hatching success was significantly correlated with incubation temperature, with nests experiencing very high temperatures exhibiting low hatching success. All nests demonstrated regular diel variation in temperature with mean daily fluctuations ranging from 0.3 degreesC to 1.4 degreesC. Increase in temperature above that of the prevailing sand temperature attributed to metabolic heating was clearly demonstrated in 14 of 15 clutches, with the mean level of metabolic heating of all nests being 0.4 degreesC. However, the level of metabolic heating varied markedly throughout the incubation period with levels being significantly higher in the final third of incubation. Incubation duration was found to be significantly correlated to both the mean temperature of nests throughout the incubation period and during the middle third of incubation. The relationship between incubation duration and mean incubation temperature was used to estimate mean incubation temperatures at most major nesting sites throughout the Mediterranean from available data on incubation durations, showing that mean incubation temperature is likely to be above 29.0 degreesC at most sites in most seasons. (C) 2001 Elsevier Science B.V. All rights reserved.

A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.

Supplementary feeding increases Common Buzzard Buteo buteo productivity but only in poor-quality habitat

Temporal heterogeneity in the effects of food supply during the breeding season on the productivity of the Common Buzzard Buteo buteo was investigated in a supplementary feeding experiment. Pairs were fed artificially (1) before egg-laying, (2) after chicks hatched and (3) continuously throughout the season, and compared with (4) unfed controls. Pairs fed before egg-laying had marginally larger clutches than those not fed, but lay date, egg volume and weight, brood size and hatching success were unaffected. Territorial quality had far greater effects, with pairs nesting in low-quality habitats (bog, scrub and semi-natural grassland) laying later and having lower hatching success, smaller broods and fewer fledglings than those in more productive agricultural landscapes. Supplementary feeding after egg hatching neutralized the negative effect of poor habitat, resulting in fed birds having significantly more fledglings. This study emphasizes the importance of food availability when provisioning chicks in suboptimal habitats and has implications for the success of diversionary feeding in reducing game losses to Buzzards.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.