The complete mitochondrial genome of the leafminer Liriomyza sativae (Diptera: Agromyzidae): great difference in the A+T-rich region compared to Liriomyza trifolii

The complete mitochondrial genome sequence of Liriomyza sativae Blanchard (15,551 bp) was determined and analyzed in this study. The circular genome contained 37 genes including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and an A + T-rich region. The initiation codons of COI and ND1 were ‘ATCA’ and ‘GTG’, respectively. ND2 gene used the truncated termination codon ‘T’. All the tRNA genes had the typical cloverleaf secondary structures except for tRNA^Ser(AGN) gene, which was found with the absence of a DHU arm. In addition, a tRNA-like secondary structure (tRNA^Met) was found in the A + T-rich region. The great difference was that the length of L. sativae A + T-rich region was 597 bp shorter than that of Liriomyza trifolii (Burgess). Meanwhile, some minor differences such as ‘TATA’ block were also observed in L. sativae in contrast to ‘TACA’ block in L. trifolii. There were also some essential structure elements such as ‘TATA’ block, ‘G(A)_nT’ block, poly-T stretch and stem-and-loop structure in the A + T-rich region of L. sativae mitochondrial genome.     

MRPS27 is a pentatricopeptide repeat domain protein required for the translation of mitochondrially encoded proteins

Mammalian pentatricopeptide repeat domain (PPR) proteins are involved in regulation of mitochondrial RNA metabolism and translation and are required for mitochondrial function. We investigated an uncharacterised PPR protein, the supernumerary mitochondrial ribosomal protein of the small subunit 27 (MRPS27), and show that it associates with the 12S rRNA and tRNA^Glu, however it does not affect their abundance. We found that MRPS27 is not required for mitochondrial RNA processing or the stability of the small ribosomal subunit. However, MRPS27 is required for mitochondrial protein synthesis and its knockdown causes decreased abundance in respiratory complexes and cytochrome c oxidase activity.

Structured summary of protein interactions

MRPS27 and MRPS15 colocalize by cosedimentation through density gradient (View Interaction)     

Predation activity of two winter-active spiders (Araneae: Anyphaenidae,Philodromidae)

Anyphaena accentuata and Philodromus spp. are cold adapted and winter-active spider species. Their predation activity was investigated at constant temperatures between –4 and 30 °C. The lower temperature threshold for Anyphaena was –3.7 °C, while that of Philodromus was –1.2 °C. At 1 °C the latency to capture and prey consumption was significantly shorter in Anyphaena than in Philodromus. The capture rate increased with temperature and was maximal at 15 °C in Anyphaena and at 30 °C in Philodromus. At 30 °C, the latency to the capture was significantly shorter in Philodromus than in Anyphaena whose mortality significantly increased.     

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis : Application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved.     

The complete mitochondrial genome of Taeniogonalos taihorina (Bischoff) (Hymenoptera: Trigonalyidae) reveals a novel gene rearrangement pattern in the Hymenoptera

The family Trigonalyidae is considered to be one of the most basal lineages in the suborder Apocrita of Hymenoptera. Here, we determine the first complete mitochondrial genome of the Trigonalyidae, from the species Taeniogonalos taihorina (Bischoff, 1914). This mitochondrial genome is 15,927 bp long, with a high A + T-content of 84.60%. It contains all of the 37 typical animal mitochondrial genes and an A + T-rich region. The orders and directions of all genes are different from those of previously reported hymenopteran mitochondrial genomes. Eight tRNA genes, three protein-coding genes and the A + T-rich region were rearranged, with the dominant gene rearrangement events being translocation and local inversion. The arrangements of three tRNA clusters, trnY–trnM–trnI–trnQ, trnW–trnL2–trnC, and trnH–trnA–trnR–trnN–trnS–trnE–trnF, and the position of the cox1 gene, are novel to the Hymenoptera, even the insects. Six long intergenic spacers are present in the genome. The secondary structures of the RNA genes are normal, except for trnS2, in which the D-stem pairing is absent.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Analysis of three leafminers' complete mitochondrial genomes

Liriomyza trifolii (Burgess), Liriomyza huidobrensis (Blanchard), and Liriomyza bryoniae (Kaltenbach), are three closely related and economically important leafminer pests in the world. This study examined the complete mitochondrial genomes of L. trifolii, L. huidobrensis and L. bryoniae, which were 16141 bp, 16236 bp and 16183 bp in length, respectively. All of them displayed 37 typical animal mitochondrial genes and an A + T-rich region. The genomes were highly compact with only 60–68 bp of non-coding intergenic spacer. However, considerable differences in the A + T-rich region were detected among the three species. Results of this study also showed the two ribosomal RNA genes of the three species had very limited variable sites and thus should not provide much information in the study of population genetics of these species. Data generated from three leafminers' complete mitochondrial genomes should provide valuable information in studying phylogeny of Diptera, and developing genetic markers for species identification in leafminers.     

Characterization of the complete mitochondrial genome of Tryporyza incertulas, in comparison with seven other Pyraloidea moths

The complete 15,223-bp mitochondrial genome (mitogenome) of Tryporyza incertulas (Walker) (Lepidoptera: Pyraloidea: Crambidae) was determined, characterized and compared with seven other species of superfamily Pyraloidea. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. Compared with other moths of Pyraloidea, the A + T biased (77.0%) of T. incertulas was the lowest. Eleven protein-coding genes (PCGs) utilized the standard ATN, but cox1 used CGA and nad4 used AAT as the initiation codons. Ten protein-coding genes had the common stop codon TAA, except nad3 having TAG as the stop codon, and cox2, nad4 using T, TA as the incomplete stop codons, respectively. All of the tRNA genes had typical cloverleaf secondary structures except trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. There was a spacer between trnQ and nad2, which was common in Lepidoptera moths. A 6-bp motif ‘ATACTA’ between trnS2(UCN) and nad1, a 7-bp motif “AGC(T)CTTA” between trnW and trnC and a 6-bp motif “ATGATA” of overlapping region between atp8 and atp6 were found in Pyraloidea moths. The A + T-rich region contained an ‘ATAGT(A)’-like motif followed by a poly-T stretch. In addition, two potential stem-loop structures, a duplicated 19-bp repeat element, and two microsatellites ‘(TA)₁₂’ and ‘(TA)₉’ were observed in the A + T-rich region of T. incertulas mitogenome. Finally, the phylogenetic relationships of Pyraloidea species were constructed based on amino acid sequences of 13 PCGs of mitogenomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These molecular-based phylogenies supported the morphological classification on relationships within Pyraloidea species.     

Temperature-induced oviposition in the brachyuran crab Cancer setosus along a latitudinal cline: Aquaria experiments and analysis of field-data

Ovigerous females of Cancer setosus are present year-round throughout most of its wide range along the Peruvian/Chilean Pacific coast (2°S-46°S). However, their number of egg-masses produced per year remains speculative and as such has neither been considered in latitudinal comparisons of reproduction, nor for its fisheries management. In order to reveal the effect of temperature on egg-mass production and egg-development, female C. setosus were held in through-flow aquaria under natural seasonal temperature conditions (16-23 °C) in Antofagasta (23°S), Northern Chile (05/2005-03/2006; 10 months), and at three constant temperatures (12, 16, 19 °C) in Puerto Montt (41°S), Central Southern Chile (09/2006-02/2007; 5 months). Female crabs uniformly produced up to 3 viable egg-masses within 4 1/2 months in Antofagasta and in Puerto Montt (at 19 °C). The second egg-mass was observed 62.5 days (± 7.6; N = 7) after the oviposition of the first clutch and a third egg-mass followed 73.5 days (± 12.5; N = 11) later in Antofagasta (at 16-23 °C). Comparably, a second oviposition took place 64.4 days (± 9.8, N = 5) after the first clutch and a third, 67.0 days (± 2.8, N = 2), thereafter, at 19 °C in Puerto Montt. At the two lower temperatures (16 and 12 °C) in Puerto Montt a second egg-mass was extruded after 82.8 days (± 28.9; N = 4) and 137 days (N = 1), respectively. The duration of egg-development from oviposition until larval hatching decreased from 65 days at 12.5 °C to 22.7 days at the observed upper temperature threshold of 22 °C. Based on the derived relationship between temperature and the duration of egg-development (y = 239.3175e^{− 0.107x}; N = 21, r² = 0.83) and data on monthly percentages of ovigerous females from field studies, the annual number of egg-masses of C. setosus was calculated. This analysis revealed an annual output of about one egg-mass close to the species northern and southern distributional limits in Casma (9°S) and Ancud (43°S), respectively, while at Coquimbo (29°S) about two and in Concepción (36°S) more than 3 egg-masses are produced per year.     

Functional screening of a novel Δ15 fatty acid desaturase from the coccolithophorid Emiliania huxleyi

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25 °C, E. huxleyi produces mainly 14:0, 18:4n − 3, 18:5n − 3 and 22:6n − 3. When the cells were transferred from 25 °C to 15 °C, the amount of unsaturated fatty acids, i.e. 18:1n − 9, 18:3n − 3 and 18:5n − 3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ?15 fatty acid desaturase, EhDES15, involved in the production of n − 3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ?6 desaturase-like domain, but it does not contain a cytochrome b₅ domain nor typical His-boxes found in ?15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n − 3 fatty acid species, which are produced at low levels in wild-type cells grown at 30 °C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ?15 desaturases. These results suggested the gene encodes a novel ?15 desaturase responsible for the synthesis of 18:3n − 3 from 18:2n − 6 in E. huxleyi.     

Enzymatic hydrolysis of cellulose by the cellobiohydrolase domain of CelB from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus

The celB gene of Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli to create a recombinant biocatalyst for hydrolyzing lignocellulosic biomass at high temperature. The GH5 domain of CelB hydrolyzed 4-nitrophenyl-β-d-cellobioside and carboxymethyl cellulose with optimum activity at pH 4.7-5.5 and 80 °C. The recombinant GH5 and CBM3-GH5 constructs were both stable at 80 °C with half-lives of 23 h and 39 h, respectively, and retained >94% activity after 48 h at 70 °C. Enzymatic hydrolysis of corn stover and cellulose pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate showed that GH5 and CBM3-GH5 primarily produce cellobiose, with product yields for CBM3-GH5 being 1.2- to 2-fold higher than those for GH5. Confocal microscopy of bound protein on cellulose confirmed tighter binding of CBM3-GH5 to cellulose than GH5, indicating that the enhancement of enzymatic activity on solid substrates may be due to the substrate binding activity of CBM3 domain.     

Multigenerational analysis of temperature and salinity variability affects on metabolic rate,generation time,and acute thermal and salinity tolerance in Daphnia pulex

Climate change, sea level rise, and human freshwater demands are predicted to result in elevated temperature and salinity variability in upper estuarine ecosystems. Increasing levels of environmental stresses are known to induce the cellular stress response (CSR). Energy for the CSR may be provided by an elevated overall metabolic rate. However, if metabolic rate is constant or lower under elevated stress, energy for the CSR is taken from other physiological processes, such as growth or reproduction. This study investigated the examined energetic responses to the combination of temperature and salinity variability during a multigenerational exposure of partheogenetically reproducing Daphnia pulex. We raised D. pulex in an orthogonal combination of daily fluctuations in temperature (15, 15–25, 15–30 °C) and salinity (0, 0–2, 0–5). Initially metabolic rates were lower under all variable temperature and variable salinity treatments. By the 6th generation there was little metabolic variation among low and intermediate temperature and salinity treatments, but metabolic suppression persisted at the most extreme salinity. When grown in the control condition for the 6th generation, metabolic suppression was only observed in D. pulex from the most extreme condition (15–30 °C, 0–5 salinity). Generation time was influenced by acclimation temperature but not salinity and was quickest in specimens reared at 15–25 °C, likely due to Q₁₀ effects at temperatures closer to the optima for D. pulex, and slowest in specimens reared at 15–30 °C, which may have reflected elevated CSR. Acute tolerance to temperature (LT₅₀) and salinity (LC₅₀) were both highest in D. pulex acclimated to 15–30 °C and salinity 0. LT₅₀ and LC₅₀ increased with increasing salinity in specimens raised at 15 °C and 15–25 °C, but decreased with increasing salinity in specimens raised at 15–30 °C. Thus, increasing temperature confers cross-tolerance to salinity stress, but the directionality of synergistic effects of temperature and salinity depend on the degree of environmental variability. Overall, the results of our study suggest that temperature is a stronger determinant of metabolism, growth, and tolerance thresholds, and assessment of the ecological impacts of environmental change requires explicit information regarding the degree of environmental variability.