A novel incubator to simulate the natural thermal environment of sea turtle eggs

A novel sea turtle egg incubator was developed in which the heating element is placed above the clutch, which more closely simulates solar heating in nature. An electronic thermometer in conjunction with a thermostat located in sand beneath a heater plate was used to obtain the desired temperature in the placed eggs, as compared to previous methods of controlling global temperature within the interior of a chamber. To test the new incubator, Lepidochelys olivacea eggs were incubated under different thermal conditions in order to identify the temperature-dependent sex determination (TSD) period more precisely. Four incubation experiments were designed to test the performance of the incubator where the temperature was lowered from 32 to 28 °C during 60 h and then reestablished at 32 °C until hatching occurred. A significant mean hatching success rate of 89.6% was obtained for all the experiments. The main result from these preliminary findings was that the sex determination period to produce males was reduced from 15 (days 15–30) to eight days (days 19–27). Overall, the incubator provides precise control and simulates a natural thermal environment that may improve control of TSD in sea turtles.     

Regeneration of lost siphon tissues in the tellinacean bivalve Nuttallia olivacea

The inhalant siphon of the tellinacean bivalve Nuttallia olivacea is an important prey item for juvenile stone flounder Platichthys bicoloratus in estuaries in Japan. We examined quantitative siphon regeneration of N. olivacea in rearing experiments of siphon-removed bivalves (> 30 mm shell length) both in the laboratory and in their natural habitat. Under laboratory conditions, siphon-removed bivalves regenerated lost tissues quantitatively at 15 and 25 °C 1 mo after siphon removal, although regeneration was incomplete. A 3-mo caging experiment in the field showed that great regeneration occurred in siphon-removed bivalves. However, the siphon weight of removed bivalves was significantly smaller than that of non-amputated bivalves, suggesting the incomplete regeneration. In a 1-mo caging experiment, bivalves that had approximately 15% of their siphons amputated were selected at some intervals to illustrate the quantitative regeneration process. Estimated daily siphon production was remarkably high only a few days after amputation. It decreased greatly thereafter, but regeneration was not completed within 30 d. These results indicate that bivalves regenerate siphons rapidly just after losing siphon tissues and then regeneration is slowed down before it is completed.     

Metabolic heating in Mediterranean loggerhead sea turtle clutches

Offspring sex ratio is an important demographic parameter and, given its determination by incubation temperature in sea turtles, might be a key factor for their conservation under climate warming. An appealing approach to estimate hatchling sex ratios is to measure sand temperatures at nest depth and deduce hatchling sex ratios from a beforehand-established relationship of hatchling sex ratio and sand temperature. Such estimates will only be accurate though if metabolic heat produced by the embryos is considered. Judging whether metabolic heating has a potential effect on hatchling sex ratios without actually measuring temperature within clutches would greatly facilitate monitoring protocols. We tested for a relationship between the amount of metabolic heating and the number of developed embryos as well as clutch size in the largest known loggerhead sea turtle (Caretta caretta) population of the Mediterranean on Zakynthos (Greece). Temperatures were measured within 20 nests as well as at a reference site in the sand at nest depth. Metabolic heating was detected, but only during the last third of the incubation period did nests heat up considerably (1.6 °C on average) above the temperature of the surrounding sand. During the middle third of incubation, when sex is determined, the amount of metabolic heating was negligible. The amount of metabolic heating during the last third of the incubation duration was significantly correlated to the number of offspring developed to at least about 75% of incubation duration. This factor explained nearly 50% of variation in metabolic heating. Metabolic heating was also significantly correlated to clutch size. Given that clutch size within the Mediterranean is largest in Zakynthos loggerheads, we conclude that metabolic heating can be ignored in the estimate of hatchling sex ratios in Mediterranean loggerhead populations. These results thus provide the basis for a feasible monitoring of hatchling sex ratios in the loggerhead sea turtle in the Mediterranean.     

Interactive effects of sublethal predation and body size on siphon production of the bivalve Nuttallia olivacea

This paper is intended to reveal effects of siphon cropping on siphon production of a tellinacean bivalve Nuttallia olivacea, an important prey for juvenile stone flounder. We carried out a two-way field experiment in which bivalves of three treatments (3-times siphon removal, 1-time removal, no removal) and four size classes (9-50 mm shell length) were caged and placed in the field for 3 mo. Growth of repeatedly-removed bivalves was inhibited, indicating reduced siphon growth (natural increase of siphon size according to somatic growth). However, siphon production of removed bivalves was larger than non-removed bivalves, possibly because of siphon regeneration. Juveniles (N. olivacea < 20 mm shell length) showed high growth performance. Their siphon growth was greater than their siphon regeneration. In all bivalves except juveniles, siphon regeneration was greater than siphon growth and engendered high siphon production. Siphon growth was dependent on bivalve size and was only slightly reduced by siphon loss, but siphon regeneration seemed to be dependent mostly on the extent of siphon loss. Greater siphon removal enhanced larger siphon production. These results indicate that intensive siphon cropping by juvenile stone flounder induces high siphon production without serious impact on N. olivacea.     

Development and characterization of ten novel microsatellite markers from olive ridley sea turtle (Lepidochelys olivacea)

Olive ridleys, one of the widely distributed marine turtle species has undergone declines in recent years due to multiple anthropogenic factors warranting conservation efforts for which assessment of genetic variability in existing populations become critical. Here we describe development of ten new microsatellite markers from a short sequence repeat-enriched partial genomic DNA library, which are found to be highly informative for genetic studies. Eight of these markers when tested on 83 olive ridley turtles revealed high allelic diversity (4–27 alleles per marker), and high observed and expected heterozygosity estimates that ranged from 0.29 to 0.82 and 0.62 to 0.94, respectively. Two microsatellites were monomorphic in the tested olive ridley samples, but were found to be informative/polymorphic when tested on related marine turtle species. More importantly, nine of the new markers showed robust cross-species amplifications in three related species Dermochelys coriacea, Chelonia mydas and Eretmochelys imbricata. Thus, this study describes ten new microsatellite markers and also demonstrates their potential as efficient genetic markers in studies related to parentage analysis, population structure, phylogeography and species relationships of olive ridleys and other marine turtle species.     

Metabolic thermogenesis and evaporative water loss in the Hwamei Garrulax canorus

Basal metabolic rate (BMR) is thought to be a major hub in the network of physiological mechanisms connecting life history traits. Evaporative water loss (EWL) is a physiological indicator that is widely used to measure water relations in inter- or intraspecific studies of birds in different environments. In this study, we examined the physiological responses of summer-acclimatized Hwamei Garrulax canorus to temperature by measuring their body temperature (T_b), metabolic rate (MR) and EWL at ambient temperatures (T_a) between 5 and 40 °C. Overall, we found that mean body temperature was 42.4 °C and average minimum thermal conductance (C) was 0.15 ml O₂ g⁻¹ h⁻¹ °C⁻¹ measured between 5 and 20 °C. The thermal neutral zone (TNZ) was 31.8–35.3 °C and BMR was 181.83 ml O₂ h⁻¹. Below the lower critical temperature, MR increased linearly with decreasing T_a according to the relationship: MR (ml O₂ h⁻¹)=266.59–2.66 T_a. At T_as above the upper critical temperature, MR increased with T_a according to the relationship: MR (ml O₂ h⁻¹)=−271.26+12.85 T_a. EWL increased with T_a according to the relationship: EWL (mg H₂O h⁻¹)=−19.16+12.64 T_a and exceeded metabolic water production at T_a>14.0 °C. The high T_b and thermal conductance, low BMR, narrow TNZ, and high evaporative water production/metabolic water production (EWP/MWP) ratio in the Hwamei are consistent with the idea that this species is adapted to warm, mesic climates, where metabolic thermogenesis and water conservation are not strong selective pressures.     

Embryonic development rate and hatchling phenotypes in the Chinese three-keeled pond turtle (Chinemys reevesii): The influence of fluctuating temperature versus constant temperature

Although the effects of constant temperatures on hatchling traits have been extensively studied in reptiles, the effects of fluctuating temperatures remain poorly understood. Eggs of the Chinese three-keeled pond turtle (Chinemys reevesii) were incubated at a constant temperatures (28 °C) and two fluctuating temperatures (28±3 °C and 28±6 °C) to test for the influence of thermal environment on incubation duration, hatchling traits, and post-hatching growth. Incubation duration was shorter at constant temperature than at fluctuating temperatures. The sex ratio of hatchlings varied among temperature treatments, with more females from 28±6 °C than from 28 °C. The size and mass were greater for hatchlings from a constant temperature than from fluctuating ones, but this difference in body size disappeared when the hatchlings were 3 months old. In addition, the swimming ability, survival, and growth of hatchlings from fluctuating temperatures did not differ from those of hatchlings from constant temperature, when they were kept at an artificial environment without food scarcity or predation. Therefore, the thermal environments with various temperature fluctuations used in this study do not significantly affect fitness-related hatchling traits in this species.     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of l-threonine

l-threonine is an essential amino acid for mammals and as such has a wide and expanding application in industry with a fast growing market demand. The major method of production of l-threonine is microbial fermentation. To optimize l-threonine production the fundamental solution is to develop robust microbial strains with high productivity and stability. Metabolic engineering provides an effective alternative to the random mutation for strain development. In this review, the updated information on genetics and molecular mechanisms for regulation of l-threonine pathways in Escherichia coli and Corynebacterium glutamicum are summarized, including l-threonine biosynthesis, intracellular consumption and trans-membrane export. Upon such knowledge, genetically defined l-threonine producing strains have been successfully constructed, some of which have already achieved the productivity of industrial producing strains. Furthermore, strategies for strain construction are proposed and potential problems are identified and discussed. Finally, the outlook for future strategies to construct industrially advantageous strains with respect to recent advances in biology has been considered.     

Metabolic alterations in the hamster co-infected with Schistosoma japonicum and Necator americanus

Co-infection with hookworm and schistosomes is a common phenomenon in sub-Saharan Africa, as well as in parts of South America and southeast Asia. As a first step towards understanding the metabolic response of a hookworm-schistosome co-infection in humans, we investigated the metabolic consequences of co-infection in an animal model, using a nuclear magnetic resonance (NMR)-based metabolic profiling technique, combined with multivariate statistical analysis. Urine and serum samples were obtained from hamsters experimentally infected with 250 Necator americanus infective L₃ and 100 Schistosoma japonicum cercariae simultaneously. In the co-infection model, similar worm burdens were observed as reported for single infection models, whereas metabolic profiles of co-infection represented a combination of the altered metabolite profiles induced by single infections with these two parasites. Consistent differences in metabolic profiles between the co-infected and non-infected control hamsters were observed from 4 weeks p.i. onwards. The predominant metabolic alterations in co-infected hamsters consisted of depletion of amino acids, tricarboxylic acid cycle intermediates (e.g. citrate and succinate) and glucose. Moreover, alterations of a series of gut microbial-related metabolites, such as decreased levels of hippurate, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid and trimethylamine-N-oxide, and increased concentrations of 4-cresol glucuronide and phenylacetylglycine were associated with co-infection. Our results provide a first step towards understanding the metabolic response of an animal host to multiple parasitic infections.     

Description of Idiomarina insulisalsae sp. nov., isolated from the soil of a sea salt evaporation pond,proposal to transfer the species of the genus Pseudidiomarina to the genus Idiomarina and emended description of the genus Idiomarina

A halophilic, aerobic Gram-negative bacterium, designated strain CVS-6^T, was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. Phylogenetic analysis of the 16S rRNA gene sequence revealed a clear affiliation of the organism with members of the family Idiomarinaceae. Sequence similarities between CVS-6^T and the type strains of the species of the genera Pseudidiomarina and Idiomarina ranged from 93.7% to 96.9%. The major isoprenoid quinone was ubiquinone 8 (Q-8). The major cellular fatty acids were 15:0 iso (21.8%), 17:0 iso (12.5%), 17:1 iso ω9c (10.7%), and 16:1 ω7c (10.6%). The DNA G+C content was 51.6 mol%. The species represented by strain CVS-6^T could be distinguished from the species of the genera Pseudidiomarina and Idiomarina; however, it was not possible to distinguish both genera from each other using the phenotypic or chemotaxonomic characteristics examined. Consequently, we propose that the species classified in the genus Pseudidiomarina should be transferred to the genus Idiomarina. We also propose that, on the basis of physiological and biochemical characteristics, strain CVS-6^T (=LMG 23123=CIP 108836) represents a new species which we name Idiomarina insulisalsae.     

Metabolic engineering of Escherichia coli: A sustainable industrial platform for bio-based chemical production

In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been significantly promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids, amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted. Moreover, this review presents guidelines for future developments in the bio-based chemical production using E. coli as an industrial platform.     

Postingestive sorting of living and heat-killed Chlorella within the sea scallop, Placopecten magellanicus (Gmelin)

Suspension-feeding bivalves may enhance the energy value of their food supply by sorting particles both before and after ingestion. Previous research has indicated that the sea scallop (Placopecten magellanicus (Gmelin) (Mollusca: Bivalvia)) is capable of sorting particles within the gut both on the basis of physical properties (particle size and density) as well as chemical properties. In this study, the ability of the sea scallop to sort living from dead material solely on the basis of chemical properties was tested. The microalga Chlorella (Chlorophyta: Chlorophyceae) was chosen as the test particle because its thick cell wall remains physically intact following heat treatment, while its carbon, nitrogen, and chlorophyll a content declines. Scallops were fed a mixture of radiolabelled live and heat-killed Chlorella. We demonstrate that P. magellanicus can distinguish between living and dead algae, retaining live Chlorella cells longer than heat-killed cells. This ability to detect the subtle chemical differences between living algal material and detrital material would enhance the digestive efficiency of this species by reducing the amount of energy expended, digesting poor-quality materials. This paper presents the first study of the ability of a bivalve to distinguish between two physically identical but nutritionally different forms of the same species of microalgae.     

Oxygen supply limits the heat tolerance of lizard embryos

The mechanisms that set the thermal limits to life remain uncertain. Classically, researchers thought that heating kills by disrupting the structures of proteins or membranes, but an alternative hypothesis focuses on the demand for oxygen relative to its supply. We evaluated this alternative hypothesis by comparing the lethal temperature for lizard embryos developing at oxygen concentrations of 10–30%. Embryos exposed to normoxia and hyperoxia survived to higher temperatures than those exposed to hypoxia, suggesting that oxygen limitation sets the thermal maximum. As all animals pass through an embryonic stage where respiratory and cardiovascular systems must develop, oxygen limitation may limit the thermal niches of terrestrial animals as well as aquatic ones.     

Summer activity pattern and Field Metabolic Rate of adult male sea otters (Enhydra lutris) in a soft sediment habitat in Alaska

Individual follows and instantaneous sampling were used to examine the behavior of adult male sea otters (Enhydra lutris) in Simpson Bay, Prince William Sound, Alaska, during the summer (May to August) of 2005 and 2006. Six behaviors (foraging, grooming, interacting with other otters, patrolling, resting, and swimming at the surface) were observed during four time periods (dawn, day, dusk, and night) to create 24-h activity budgets. Adult male sea otters were observed during 190 focal follows, representing 98 h of observation. Male otters allocated 27% of their time (over a 24-h period) to resting, 26% to swimming, 19% to grooming, 14% foraging, 9% to patrolling and 5% to interacting with other otters. Field Metabolic Rate (FMR) was estimated by combining the energetic costs for foraging, grooming, resting, and swimming behavior of captive otters from Yeates et al. (2007) with our activity budgets. Our study considered ‘patrolling’ to be energetically similar to ‘swimming’ and therefore the two categories were combined. This combined category accounted for the greatest percentage (43%) of energy expended each day followed by grooming (23%), resting (15%), feeding (13%) and other (5%). The estimated weight specific FMR for all activities was 686.7 kJ day^− 1 kg^− 1 and the total FMR was 19.04 MJ day^− 1. The FMR was 6.6 times the resting metabolic rate and 2.2 times greater than the allometric prediction for terrestrial mammals of similar size but similar to other marine mammals. With a peak summer sea otter density in 5.6 otters km^− 2, the low percentage of time spent foraging (even after correction for possible sampling biases) indicates that Simpson Bay is still below equilibrium density.     

Effects of acclimation and egg-incubation temperature on selected temperature by hatchling western painted turtles (Chrysemys picta bellii)

The ability of hatchling turtles to detect environmental temperature differences and to effectively select preferred temperature is a function that critically impacts survival. In some turtle species, temperature preference may be influenced by embryonic and post-hatching conditions, such as egg-incubation and acclimation temperature. We tested for effects of embryonic incubation temperature (27.5 °C, 30 °C) and acclimation temperature (20 °C, 25 °C) on the selected temperature and movement patterns of 32 Chrysemys picta bellii (Reptilia: Emydidae) hatchlings in an aquatic thermal gradient of 14-34 °C and in single-temperature (20 °C, 25 °C) control tests. Among 10-11 month old hatchlings, acclimation temperature and egg-incubation temperature influenced temperature selection and movement patterns. Acclimation temperature affected activity and movement: in thermal gradient and single-temperature control tests, 25 °C-acclimated turtles relocated between chambers significantly more frequently than individuals acclimated to 20 °C. Acclimation temperature also affected temperature selection: 20 °C-acclimated turtles selected a specific temperature during gradient tests, but 25 °C-acclimated turtles did not. Among 20 °C-acclimated turtles, egg-incubation temperature was inversely related to selected temperature: hatchling turtles incubated at 27.5 °C selected the warmest temperature available (34 °C); individuals incubated at 30 °C selected the coldest temperature (14 °C). These results suggest that interactions of environmental conditions may influence post-hatching thermoregulatory behavior in C. picta bellii, a factor that ultimately affects fitness.     

Metabolic classification of South American Ilex species by NMR-based metabolomics

The genus Ilex to which mate (Ilex paraguariensis) belongs, consists of more than 500 species. A wide range of metabolites including saponins and phenylpropanoids has been reported from Ilex species. However, despite the previous works on the Ilex metabolites, the metabolic similarities between species which can be used for chemotaxonomy of the species are not clear yet. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics was applied to the classification of 11 South American Ilex species, namely, Ilex argentina, Ilex brasiliensis, Ilex brevicuspis, Ilex dumosa var. dumosa, I. dumosa var. guaranina, Ilex integerrima, Ilex microdonta, I. paraguariensis var. paraguariensis, Ilex pseudobuxus, Ilex taubertiana, and Ilex theezans. ¹H NMR combined with principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) showed a clear separation between species and resulted in four groups based on metabolomic similarities. The signal congestion of ¹H NMR spectra was overcome by the implementation of two-dimensional (2D)-J-resolved and heteronuclear single quantum coherence (HSQC). From the results obtained by 1D- and 2D-NMR-based metabolomics it was concluded that species included in group A (I. paraguariensis) were metabolically characterized by a higher amount of xanthines, and phenolics including phenylpropanoids and flavonoids; group B (I. dumosa var. dumosa and I. dumosa var. guaranina) with oleanane type saponins; group C (I. brasiliensis, I. integerrima, I. pseudobuxus and I. theezans) with arbutin and dicaffeoylquinic acids, and group D (I. argentina, I. brevicuspis, I. microdonta and I. taubertiana) with the highest level of ursane-type saponins. Clear metabolomic discrimination of Ilex species and varieties in this study makes the chemotaxonomic classification of Ilex species possible.     

Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae)

Ambrosia beetles (Coleoptera: Scolytinae) are associated with strictly entomochoric and mutualistic fungi. We studied the mycobiota associated with Scolytodes unipunctatus, ambrosia beetles that infest Cecropia trees in Central America. Isolates were characterized using morphology and rDNA sequences (ITS region, LSU, and SSU rDNA). Four species are described here: Raffaelea scolytodis sp. nov. (Ophiostomatales), Gondwanamyces scolytodis sp. nov., Custingophora cecropiae sp. nov., and Graphium sp. (Microascales). The genus Custingophora is emended to include Knoxdaviesia anamorphs of Gondwanamyces based on uniformity of DNA sequences and phenotype.