Geographic variation in the time‐frequency characteristics of high‐frequency whistles produced by killer whales (Orcinus orca)

Investigating intraspecific variation in acoustic signals can indicate the extent of isolation and divergence between populations and adaptations to local environments. Here we analyze the variation in killer whale high‐frequency (>17 kHz) whistles recorded off Norway, Iceland, and in the North Pacific. We used a combination of methods including multivariate comparisons of spectral and temporal parameters and categorization of contours to types. Our results show that spectral and temporal characteristics of high‐frequency whistles recorded in the North Pacific show significant differences from whistles recorded in the Northeast Atlantic, being generally stereotyped, lower in frequency, and slightly longer in duration. Most high‐frequency whistles from the North Pacific were downsweeps, whereas this was one of the least common types recorded in the Northeast Atlantic. The repertoire of whistles recorded in Norway was similar to Iceland, but whistles produced in Norway had significantly lower maximum frequency and frequency range. Most methods were able to discriminate between whistles of the North Pacific and the Northeast Atlantic, but were unable to consistently distinguish whistles from Iceland and Norway. This suggests that macro‐ and microgeographic differences in high‐frequency whistles of killer whales may reflect historical geographic isolation between ocean basins and more recent divergence between adjacent populations.     

Life history and population dynamics of southern Alaska resident killer whales (Orcinus orca)

Resident (fish eating) killer whales (Orcinus orca) in the North Pacific have been the subject of long‐term studies in several geographical regions. The current study examines population parameters in the southern Alaska resident population from 1984 to 2010 and develops a population model. The southern Alaska resident population ranges from southeastern Alaska through the Kodiak archipelago and contains over 700 individuals. We follow the life histories of 343 identifiable whales in 10 pods from two clans born before and during the study. Population parameters were comparable to those of the British Columbia northern resident population during the 1970s and 1980s, except that age of maturity was approximately one year earlier. The average annual rate of increase was slightly higher in Alaska (3.5%) than for the British Columbia northern residents (2.9%) and probably represents a population at r‐max (maximum rate of growth). Reasons for the high growth rate in Alaska could be a recovery following past anthropogenic mortalities, or more likely, a response to increasing salmon returns in recent decades, resulting in an increase in carrying capacity. The slow maturation and low rate of reproductive response makes these whales slow to recover from natural or anthropogenic catastrophes.     

Spatial and temporal analysis of killer whale (Orcinus orca) strandings in the North Pacific Ocean and the benefits of a coordinated stranding response protocol

Killer whales (Orcinus orca) are widely distributed throughout the world's oceans, yet little has been documented about their stranding patterns. Knowledge of stranding patterns improves our ability to examine and sample carcasses and provides a foundation for understanding killer whale natural history, diet, reproduction, anthropogenic stressors, emerging diseases, and patterns of unusual mortality. We compiled published and unpublished killer whale stranding data to describe stranding patterns in the North Pacific Ocean. Between 1925 and 2011, 371 stranded killer whales were reported in Japan (20.4%), Russia (3.5%), Alaska (32.0%), British Columbia (27.4%), Washington (4.0%), Oregon (2.7%), California (5.1%), Mexico (3.8%), and Hawaii (0.8%). Strandings occurred at all times of year, but regionally specific seasonal differences were observed. Mortality and annual census data from Northern and Southern Resident populations were extrapolated to estimate that across the North Pacific, an average of 48 killer whales die annually. However, over the last two decades, an average of only 10 killer whale carcasses were recovered annually in this ocean, making each event a rare opportunity for study. Publication of a standardized killer whale necropsy protocol and dedicated funding facilitated the number of complete postmortem necropsies performed on stranded killer whales from 1.6% to 32.2% annually.     

Evidence for the functions of surface‐active behaviors in humpback whales (Megaptera novaeangliae)

As part of their social sound repertoire, migrating humpback whales (Megaptera novaeangliae) perform a large variety of surface‐active behaviors, such as breaching and repetitive slapping of the pectoral fins and tail flukes; however, little is known about what factors influence these behaviors and what their functions might be. We investigated the potential functions of surface‐active behaviors in humpback whale groups by examining the social and environmental contexts in which they occurred. Focal observations on 94 different groups of whales were collected in conjunction with continuous acoustic monitoring, and data on the social and environmental context of each group. We propose that breaching may play a role in communication between distant groups as the probability of observing this behavior decreased significantly when the nearest whale group was within 4,000 m compared to beyond 4,000 m. Involvement in group interactions, such as the splitting of a group or a group joining with other whales, was an important factor in predicting the occurrence of pectoral, fluke, and peduncle slapping, and we suggest that they play a role in close‐range or within‐group communication. This study highlights the potentially important and diverse roles of surface‐active behaviors in the communication of migrating humpback whales.     

Real‐time PCR detection of Didemnum perlucidum (Monniot, 1983) and Didemnum vexillum (Kott, 2002) in an applied routine marine biosecurity context

Prevention and early detection are well recognized as the best strategies for minimizing the risks posed by nonindigenous species (NIS) that have the potential to become marine pests. Central to this is the ability to rapidly and accurately identify the presence of NIS, often from complex environmental samples like biofouling and ballast water. Molecular tools have been increasingly applied to assist with the identification of NIS and can prove particularly useful for taxonomically difficult groups like ascidians. In this study, we have developed real‐time PCR assays suited to the specific identification of the ascidians Didemnum perlucidum and Didemnum vexillum. Despite being recognized as important global pests, this is the first time specific molecular detection methods have been developed that can support the early identification and detection of these species from a broad range of environmental sample types. These fast, robust and high‐throughput assays represent powerful tools for routine marine biosecurity surveillance, as detection and confirmation of the early presence of species could assist in the timely establishment of emergency responses and control strategies. This study applied the developed assays to confirm the ability to detect Didemnid eDNA in water samples. While previous work has focused on detection of marine larvae from water samples, the development of real‐time PCR assays specifically aimed at detecting eDNA of sessile invertebrate species in the marine environment represents a world first and a significant step forwards in applied marine biosecurity surveillance. Demonstrated success in the detection of D. perlucidum eDNA from water samples at sites where it could not be visually identified suggests value in incorporating such assays into biosecurity survey designs targeting Didemnid species.     

Non‐specific immunity and disease resistance are enhanced by the polysaccharide fraction of a marine chlorophycean macroalga in Oreochromis niloticus (Linnaeus, 1758)

Polysaccharides (PF) from marine macroalgae, Caulerpa scalpelliformis were extracted and tested for its potential immunostimulatory and disease resistance properties in fish. Five groups of Nile tilapia (n = 6), Oreochromis niloticus (Linnaeus, 1758) were intraperitoneally administered with the different doses of PF (2, 20 or 200 mg/kg body weight) or with yeast‐derived commercial immunostimulant, Macrogard^? (20 mg/kg body weight), to compare the effectiveness. An untreated control group was also maintained. A total of fifteen fibre reinforced plastic tanks (150 L, ambient temperature and light conditions) were used, with triplicate tanks for each group. Only four fish per tank (totally 12 fish from a group) were taken at random and assayed. PF enhanced all the tested non‐specific serum immune responses namely lysozyme, myeloperoxidase, antiprotease, and bactericidal activities. There was an upregulation of the genes encoding IL‐1β, lysozyme and TNF‐α in the spleen of PF injected fish as compared to the control group. In order to study the overall functional immunity, disease resistance test was conducted. Another five groups of fish (n = 10) were treated by intraperitoneal injection with different doses of PF or Macrogard^? or untreated as mentioned earlier in triplicates (30 fish per group in three tanks, totally 150 fish in 15 tanks). Seven days post treatment, fish were challenged by intraperitoneal administration of live virulent Aeromonas hydrophila. PF treated fish were protected with significant reduction in the mortality and the consequent increased relative percent survival (RPS) of 92 in the least (2 mg/kg) and middle dose (20 mg/kg) groups. The disease resistance experiment was repeated again but this time, fish were challenged 21 days post treatment that resulted in RPS of 50 for the middle dose. The results clearly show that the intraperitoneal administration of the polysaccharide fraction had a stimulating effect on the non‐specific immune responses, immune gene expression and disease resistance.     

Elemental composition of strawberry plants inoculated with the plant growth‐promoting bacterium Azospirillum brasilense REC3, assessed with scanning electron microscopy and energy dispersive X‐ray analysis

The elemental composition of strawberry plants (Fragaria ananassa cv. Macarena) inoculated with the plant growth‐promoting bacterium Azospirillum brasilense REC3, and non‐inoculated controls, was studied using scanning electron microscopy (SEM) and energy dispersive X‐ray (EDS) analysis. This allowed simultaneous semi‐quantification of different elements in a small, solid sample. Plants were inoculated and grown hydroponically in 50% or 100% Hoagland solution, corresponding to limited or optimum nutrient medium, respectively. Bacteria‐inoculated plants increased the growth index 45% and 80% compared to controls when grown in 100% and 50% Hoagland solution, respectively. Thus, inoculation with A. brasilense REC3 in a nutrient‐limited medium had the strongest effect in terms of increasing both shoot and root biomass and growth index, as already described for Azospirillum inoculated into nutrient‐poor soils. SEM‐EDS spectra and maps showed the elemental composition and relative distribution of nutrients in strawberry tissues. Leaves contained C, O, N, Na, P, K, Ca and Cu, while roots also had Si and Cl. The organic fraction (C, O and N) accounted for over 96.3% of the total chemical composition; of the mineral fraction, Na had higher accumulation in both leaves and roots. Azospirillum‐inoculated and control plants had similar elemental quantities; however, in bacteria‐inoculated roots, P was significantly increased (34.33%), which constitutes a major benefit for plant nutrition, while Cu content decreased (35.16%).     

First Report of a 16SrI‐C Phytoplasma Infecting Celery (Apium graveolens) with Stunting,Bushy Top and Phyllody in the Czech Republic

Apium graveolens L. plants showing stunting, purplish/whitening of new leaves, flower abnormalities and bushy tops were observed in South Bohemia (Czech Republic) during 2011 and 2012. Transmission electron microscopy observations showed phytoplasmas in phloem sieve tube elements of symptomatic but not healthy plants. Polymerase chain reactions with universal and group‐specific phytoplasma primers followed by restriction fragment length polymorphism analyses and sequencing of 16S rDNA enabled classification of the detected phytoplasmas into the aster yellows group, ribosomal subgroup 16SrI‐C. Identical analyses of the ribosomal protein genes rpl22 and rps3 were used for further classification and revealed affiliation of the phytoplasmas with the rpIC subgroups. This is the first report of naturally occurring clover phyllody phytoplasma in A. graveolens in both the Czech Republic and worldwide.     

Physio‐biochemical assessment and expression analysis of genes associated with drought tolerance in sugarcane (Saccharum spp. hybrids) exposed to GA3 at grand growth stage

• Drought is one of the most serious environmental factors limiting production of sugarcane worldwide. In order to assess the influence of gibberellins (GA₃) on drought and plant growth, along with associated physio‐biochemical attributes, expression of eight drought‐responsive genes were quantified and analysed.
• At grand growth stage (120 DAP) two sugarcane varieties (CoLk94184, CoPK05191) were exposed to drought by withholding irrigation. GA₃ (35 ppm) was applied using battery‐operated uniform controlled dispensing sprayer twice at 1‐week intervals on 2‐week drought‐stressed plants. Physio‐biochemical attributes including antioxidant enzyme activities were estimated following standard protocols. RT‐PCR was performed to visualise the drought‐associated gene expression patterns.
• Drought triggered a reduction in RWC and chlorophyll content but these recovered when droughted plants were exposed to GA₃. Proline content increased many fold in both varieties under stress, but decreased under the influence of GA₃. There was a mixed response of antioxidant enzyme activity, which distinctly declined after GA₃ exposure, together with a lesser reduction in dry matter content over that of control plants. With increasing stress, expression of pyrroline‐5‐carboxylase synthetase (P5CS) and betaine‐aldehyde dehydrogenase genes was observed, selectively up‐regulated in CoPK05191. Expression of proline oxidase/transporter was high in CoPK05191 but diminished along with proline content after exposure to GA_3. CoLk94184 showed no significant difference in P5CS gene expression under stress condition, whereas expression of betaine‐aldehyde dehydrogenase gene was unchanged in response to stress.
• Results demonstrated that exposure of droughted plants to GA₃ not only led to recovery of activity of drought‐associated physio‐biochemical attributes, but also minimised impact on cane dry weight and quality. Further, GA₃ application caused differential gene expression that possibly triggers increased responsiveness towards drought tolerance in sugarcane.

     

Marginal land bioethanol yield potential of four crassulacean acid metabolism candidates (Agave fourcroydes,Agave salmiana,Agave tequilana and Opuntia ficus‐indica) in Australia

The Nobel environmental productivity index (EPI) was used as a framework for the development of a predictive geospatial model to estimate the bioethanol yield potential of four crassulacean acid metabolism (CAM) candidates in Australia (Agave fourcroydes, Agave salmiana, Agave tequilana, and Opuntia ficus‐indica). GIS software was used to integrate climate datasets with titratable acidity responses to changes in photosynthetically active radiation (PAR), temperature, and water availability. Additional refinements to Nobel's approach were made to accommodate spatial and temporal fluctuations in soil water potential (ψs) as a function of soil particle size distribution and precipitation, and CO₂ uptake response to a range of day and night temperatures. A scalar factor for CO₂ persistence during periods of drought was also introduced to model the capacity of succulent species of Agave to buffer against fluctuations in ψs. Macro‐scale criteria were applied to estimate environmentally responsible (ER) bioethanol yield potential on lands that are not suitable for food production. Consideration was given to indigenous vascular plant species richness and endemism scores at ER sites of interest. The highest mean ER bioethanol yield was achieved by A. fourcroydes (μ: 3.89, max. 7.17 kL ha^‐1 yr^‐1) while the highest maximum yield was achieved by A. tequilana (μ: 3.78, max. 7.63 kL ha^‐1 yr^‐1). This research indicated the CAM pathway may produce significant yields (≥≥ 5 kL ha^‐1 yr^‐1) at ER sites totalling 57,700 km² (0.7% land area of Australia).     

Limber pine (Pinus flexilis James) genetic map constructed by exome‐seq provides insight into the evolution of disease resistance and a genomic resource for genomics‐based breeding

Limber pine ( Pinus flexilis ) is a keystone species of high‐elevation forest ecosystems of western North America, but some parts of the geographic range have high infection and mortality from the non‐native white pine blister rust caused by Cronartium ribicola . Genetic maps can provide essential knowledge for understanding genetic disease resistance as well as local adaptation to changing climates. Exome‐seq was performed to construct high‐density genetic maps in two seed families. Composite maps positioned 9612 unigenes across 12 linkage groups ( LG s). Syntenic analysis of genome structure revealed that the majority of orthologs were positional orthologous genes ( POG s) with localization on homologous LG s among conifer species. Gene ontology ( GO) enrichment analysis showed relatively fewer constraints for POG s with putative roles in adaptation to environments and relatively more conservation for POG s with roles in basic cell function and maintenance. The mapped genes included 639 nucleotide‐binding site leucine‐rich repeat genes ( NBS ‐ LRR s) , 290 receptor‐like protein kinase genes ( RLK s), and 1014 genes with potential roles in the defense response and induced systemic resistance to attack by pathogens. Orthologous loci for resistance to rust pathogens were identified and were co‐positioned with multiple members of the R gene family, revealing the evolutionary pressure acting upon them. This high‐density genetic map provides a genomic resource and practical tool for breeding and genetic conservation programs, with applications in genome‐wide association studies ( GWASs ), the characterization of functional genes underlying complex traits, and the sequencing and assembly of the full‐length genomes of limber pine and related Pinus species.     

Morphological and Molecular Redefinition of Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and Aspidisca lynceus (Müller,1773) Ehrenberg, 1859, with Reconsideration of a “Well‐known” Euplotes Ciliate,Euplotes harpa Stein, 1859 (Ciliophora,Euplotida)

We documented the morphology, infraciliature, silverline system, and molecular data of two euplotid species isolated from China, including two populations of the poorly known Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and the previously well described Aspidisca lynceus (Müller, 1773 ) Ehrenberg, 1830. Based on the information available, an improved diagnosis of Euplotes platystoma is given, including: a narrow adoral zone with 44–68 membranelles, 10 frontoventral, 5 transverse, 2 left marginal and 2 caudal cirri, 11–13 dorsal kineties with 17–25 dikinetids in the mid‐dorsal row, and dorsal silverline system of the double‐eurystomus type. The Chinese population of Aspidisca lynceus closely resembles previously described populations. Phylogenetic analyses inferred from SSU rDNA sequences show that E. platystoma is closely related with E. neapolitanus, and the internal position of A. lynceus within this genus is still not robust. A reconsideration of the “well‐known” Euplotes harpa and a comparison of all SSU rDNA sequences of E. harpa in GenBank are provided. We speculate that the sequences available from GenBank under the name of E. harpa are very likely from misidentified materials, that is, the identity of the species currently associated with the SSU rDNA of this “well‐known” form in molecular databases requires further confirmation.     

Sequential interactions of silver–silica nanocomposite (Ag–SiO2NC) with cell wall,metabolism and genetic stability of Pseudomonas aeruginosa,a multiple antibiotic‐resistant bacterium

The study was carried out to understand the effect of silver–silica nanocomposite (Ag–SiO₂NC) on the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, a multiple drug‐resistant bacterium. Bacterial sensitivity towards antibiotics and Ag–SiO₂NC was studied using standard disc diffusion and death rate assay, respectively. The effect of Ag–SiO₂NC on cell wall integrity was monitored using SDS assay and fatty acid profile analysis, while the effect on metabolism and genetic stability was assayed microscopically, using CTC viability staining and comet assay, respectively. Pseudomonas aeruginosa was found to be resistant to β‐lactamase, glycopeptidase, sulfonamide, quinolones, nitrofurantoin and macrolides classes of antibiotics. Complete mortality of the bacterium was achieved with 80 μg ml^?1 concentration of Ag–SiO₂NC. The cell wall integrity reduced with increasing time and reached a plateau of 70% in 110 min. Changes were also noticed in the proportion of fatty acids after the treatment. Inside the cytoplasm, a complete inhibition of electron transport system was achieved with 100 μg ml^?1 Ag–SiO₂NC, followed by DNA breakage. The study thus demonstrates that Ag–SiO₂NC invades the cytoplasm of the multiple drug‐resistant P. aeruginosa by impinging upon the cell wall integrity and kills the cells by interfering with electron transport chain and the genetic stability.

Significance and Impact of Study

Although the synthesis, structural characteristics and biofunction of silver nanoparticles are well understood, their application in antimicrobial therapy is still at its infancy as only a small number of microorganisms are tested to be sensitive to nanoparticles. A thorough knowledge of the mode of interaction of nanoparticles with bacteria at subcellular level is mandatory for any clinical application. The present study deals with the interactions of Ag–SiO2NC with the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, which would contribute substantially in strengthening the therapeutic applications of silver nanoparticles.