Stable Colonies Research Articles

Relationship Between the Host Plant Range of Insects and Symbiont Bacteria.

The evolution of phytophagous insects has resulted in the development of feeding specializations that are unique to this group. The majority of current research on insect palatability has concentrated on aspects of ecology and biology, with relatively little attention paid to the role of insect gut symbiotic bacteria. Symbiont bacteria have a close relationship with their insect hosts and perform a range of functions. This research aimed to investigate the relationship between insect host plant range and gut symbiotic bacteria. A synthesis of the extant literature on the intestinal commensal bacteria of monophagous, oligophagous, and polyphagous tephritids revealed no evidence of a positive correlation between the plant host range and the diversity of larval intestinal microbial species. The gut symbionts of same species were observed to exhibit discrepancies between different literature sources, which were attributed to variations in multiple environmental factors. However, following beta diversity analysis, monophagy demonstrated the lowest level of variation in intestinal commensal bacteria, while polyphagous tephritids exhibited the greatest variation in intestinal commensal bacteria community variation. In light of these findings, this study proposes the hypothesis that exclusive or closely related plant hosts provide monophagy and oligophagy with a stable core colony over long evolutionary periods. The core flora is closely associated with host adaptations in monophagous and oligophagous tephritids, including nutritional and detoxification functions. This is in contrast to polyphagy, whose dominant colony varies in different environments. Our hypothesis requires further refinement of the data on the gut commensal bacteria of monophagy and oligophagy as the number of species and samples is currently limited.

Growth kinetics and population density of a laboratory colony of the poultry red mite (Dermanyssus gallinae) established in Japan.

The poultry red mite (PRM), Dermanyssus gallinae, is a hematophagous ectoparasite that significantly threatens the poultry industry, not only through blood-feeding but also as a vector for deadly pathogens. With the growing challenge of acaricidal resistance, the demand for alternative control measures is urgent. However, effective PRM research, particularly in acaricidal efficacy and new drug discovery, hinges on the availability of reliable laboratory colonies. In this study, we successfully established a stable PRM laboratory colony, originally isolated from the field in 2021 and maintained under controlled conditions at the Research Institute for Animal Science in Biochemistry and Toxicology (RIAS). We investigated the growth kinetics and population dynamics of the laboratory colony within a Styrofoam-based maintenance box (SBMB) containing chicks. PRM propagation was tracked over 28 days, with mites collected every seven days. The average bulk weight of the mites in the trap increased from 4.3 ± 1.2 mg on day 7 to 201.4 ± 56.5 mg on day 28, despite seasonal variations, indicating optimal conditions for population growth. The collected mites spanned various blood-feeding developmental stages such as protonymph, deutonymph, and adult stages, enabling comprehensive assessments of molting and egg-laying efficiency. Our findings confirm that the laboratory colony of PRM can be stably maintained, providing a reliable source of PRMs for further experimental research aimed at advancing control strategies against this pervasive pest.

Wild black soldier flies, Hermetia illucens (Diptera: Stratiomyidae): Seasonal availability and life history traits in two common organic streams in Bangladesh

The black soldier fly (BSF) Hermetia illucens (L.) (Diptera: Stratiomyidae) is a “crown jewel” in waste management because it rapidly bioconverts organic streams into nutrient-rich biomass. Bangladesh's inefficient waste management has caused serious environmental issues due to the massive amounts of organic waste, mainly livestock manures and municipal organic waste, generated daily. We need reliable BSF to maximize our organic resources. To establish a long-term, stable BSF colony, we closely examined the seasonal abundance and life cycle features of BSF raised on municipal garbage and poultry manure in a regional natural environment in the lab. This investigation lasted two years (2019–2020). Season and raising substrates affected wild BSF egg clutches and life cycles. Organic waste dumps captured the most egg clutches in rainy seasons and the fewest in cooler months. Municipal rubbish dumps consistently produced wilder BSF egg clutches than poultry manure dumpsites. The BSF larvae grew slower in winter and faster in the rainy season, independent of substrate. Winter was optimal for adult BSF longevity in all organic streams. Winter cultivated wild BSF on two organic streams exhibited fewer egg clutches per female, shorter and lighter prepupae, and lower adult ecolosion rates than wetter months. While BSF given municipal organic wastes had the highest adult ecolosion rates, BSF fed poultry manure had more variation in prepupae length and weight, adult lifespan, and egg clutches. Larval development was faster with poultry manure BSF than with municipal organic waste. The protein and ash content of prepupae rearing on poultry manure exceeded those of prepupae on urban organic waste, although fat content was equivalent. These findings indicate that both types of organic streams may be suitable substrates for BSF larvae for trash treatment.

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages.

Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.

Evaluating risks to seabirds on the urban–coastal interface: Modelling dog attacks on little penguin populations in Tasmania

Abstract Tasmania (including offshore islands) has the largest breeding population of little penguins (Eudyptula minor) in Australia, but coastal development around nesting areas has led to habitat loss, often resulting in smaller, fragmented colonies. Colonies have also been subject to predation by introduced domestic species. This study assessed whether historical levels of domestic dog predation could threaten the viability of penguin populations. Data collected since the 1980s detailing little penguin kills by domestic dogs were used to inform the extent of individual mortality events. The potential impact of dog attacks on the viability of little penguin colonies of the sizes typically found on mainland Tasmania was explored using an age‐based population model. Simulation was conducted over decadal timescales to assess the impact of attacks at varying frequencies and intensities on small (100 birds), medium (300 birds) and large (500 birds) colonies that were stable, growing or decreasing. Results suggested that frequent and intense dog attacks markedly increased the probability of colony decline, regardless of colony size. Even large, growing penguin colonies were at risk of decline. The risk of extinction was <10% for medium and large stable colonies if no more than 15–25 penguins were killed per attack and there were no more than 20 attacks per 50 years. For small‐sized colonies, the risk of extinction was <10% if fewer than 10 attacks occurred per 50‐year period and no more than 15 birds were killed per attack. Importantly, results suggested that for small colonies especially, even low levels of predator attack could lead to colony collapse in 10–15 years. For large colonies that were already decreasing, the average time to extinction was markedly increased with additional mortalities from dogs, highlighting the need for ongoing management measures that reduce or eliminate dog attacks to help conserve this iconic species.

Neural Differentiation and spinal cord organoid generation from induced pluripotent stem cells (iPSCs) for ALS modelling and inflammatory screening.

C9orf72 genetic mutation is the most common genetic cause of ALS/FTD accompanied by abnormal protein insufficiency. Induced pluripotent stem cell (iPSC)-derived two-dimensional (2D) and three-dimensional (3D) cultures are providing new approaches. Therefore, this study established neuronal cell types and generated spinal cord organoids (SCOs) derived from C9orf72 knockdown human iPSCs to model ALS disease and screen the unrevealed phenotype. Wild-type (WT) iPSC lines from three healthy donor fibroblasts were established, and pluripotency and differentiation ability were identified by RT-PCR, immunofluorescence and flow cytometry. After infection by the lentivirus with C9orf72-targeting shRNA, stable C9-knockdown iPSC colonies were selected and differentiated into astrocytes, motor neurons and SCOs. Finally, we analyzed the extracted RNA-seq data of human C9 mutant/knockout iPSC-derived motor neurons and astrocytes from the GEO database and the inflammatory regulation-related genes in function and pathways. The expression of inflammatory factors was measured by qRT-PCR. The results showed that both WT-iPSCs and edited C9-iPSCs maintained a similar ability to differentiate into the three germ layers, astrocytes and motor neurons, forming SCOs in a 3D culture system. The constructed C9-SCOs have features of spinal cord development and multiple neuronal cell types, including sensory neurons, motor neurons, and other neurons. Based on the bioinformatics analysis, proinflammatory factors were confirmed to be upregulated in C9-iPSC-derived 2D cells and 3D cultured SCOs. The above differentiated models exhibited low C9orf72 expression and the pathological characteristics of ALS, especially neuroinflammation.

Cryopreserved ovine spermatogonial stem cells maintain stemness and colony forming ability in vitro

Objective: To assess the effect of cryopreservation on stemness and proliferation potential of sheep spermatogonial stem cells (SSCs) in vitro. Methods: Sheep testicular cells were isolated and putative SSCs were enriched by the laminin-based differential plating method. Putative SSCs were co-cultured with the Sertoli cell feeder prepared by the Datura Stramonium Agglutinin (DSA-lectin)-based method. The cultured putative SSCs were cryopreserved in Dulbecco's Modified Eagle Medium-10% fetal bovine serum mixture (DMEM-10% FBS) media containing 10% dimethyl sulfoxide (DMSO) alone or 10% DMSO plus 200 mM trehalose. Cryopreserved putative SSCs were evaluated for their proliferation potential using in vitro culture and stemness by immunocytochemistry. Finally, the transfection ability of cryopreserved putative SSCs was analyzed. Results: We isolated 91% viable testicular cells from sheep testes. The majority of the laminin enriched cells expressed the SSC related marker, ITGA6. Co-culture of sheep putative SSCs with Sertoli cell feeder resulted in the generation of stable colonies, and the expression of SSC marker was maintained after several passages. A significantly higher number of viable putative SSCs was recovered from SSCs cryopreserved in media containing 10% DMSO and 200 mM trehalose compared to 10% DMSO alone (P<0.01). Cryopreserved putative SSCs formed colonies and showed SSC marker expression similar to the non-cryopreserved putative SSCs. The appearance of green fluorescent colonies over the Sertoli cell feeder indicated that cryopreserved sheep SSCs were successfully transfected. Conclusions: Cryopreserved putative SSCs can retain their stemness, colony forming ability, and transfection efficiency in vitro. Our research may help in the effective preservation of germplasm and the generation of transgenic ovine species.

The Controversial Effect of Antibiotics on Methicillin-Sensitive S. aureus: A Comparative In Vitro Study.

Methicillin-sensitive Staphylococcus (S.) aureus (MSSA) bacteremia remains a global challenge, despite the availability of antibiotics. Primary treatments include β-lactam agents such as cefazolin and flucloxacillin. Ongoing discussions have focused on the potential synergistic effects of combining these agents with rifampicin or fosfomycin to combat infections associated with biofilm formation. Managing staphylococcal infections is challenging due to antibacterial resistance, biofilms, and S. aureus's ability to invade and replicate within host cells. Intracellular invasion shields the bacteria from antibacterial agents and the immune system, often leading to incomplete bacterial clearance and chronic infections. Additionally, S. aureus can assume a dormant phenotype, known as the small colony variant (SCV), further complicating eradication and promoting persistence. This study investigated the impact of antibiotic combinations on the persistence of S. aureus 6850 and its stable small colony variant (SCV strain JB1) focusing on intracellular survival and biofilm formation. The results from the wild-type strain 6850 demonstrate that β-lactams combined with RIF effectively eliminated biofilms and intracellular bacteria but tend to select for SCVs in planktonic culture and host cells. Higher antibiotic concentrations were associated with an increase in the zeta potential of S. aureus, suggesting reduced membrane permeability to antimicrobials. When using the stable SCV mutant strain JB1, antibiotic combinations with rifampicin successfully cleared planktonic bacteria and biofilms but failed to eradicate intracellular bacteria. Given these findings, it is reasonable to report that β-lactams combined with rifampicin represent the optimal treatment for MSSA bacteremia. However, caution is warranted when employing this treatment over an extended period, as it may elevate the risk of selecting for small colony variants (SCVs) and, consequently, promoting bacterial persistence.

Emergence of sector and spiral patterns from a two-species mutualistic cross-feeding model.

The ubiquitous existence of microbial communities marks the importance of understanding how species interact within the community to coexist and their spatial organization. We study a two-species mutualistic cross-feeding model through a stochastic cellular automaton on a square lattice using kinetic Monte Carlo simulation. Our model encapsulates the essential dynamic processes such as cell growth, and nutrient excretion, diffusion and uptake. Focusing on the interplay among nutrient diffusion and individual cell division, we discover three general classes of colony morphology: co-existing sectors, co-existing spirals, and engulfment. When the cross-feeding nutrient is widely available, either through high excretion or fast diffusion, a stable circular colony with alternating species sector emerges. When the consumer cells rely on being spatially close to the producers, we observe a stable spiral. We also see one species being engulfed by the other when species interfaces merge due to stochastic fluctuation. By tuning the diffusion rate and the growth rate, we are able to gain quantitative insights into the structures of the sectors and the spirals.

Transcriptome analysis of mouse male germline stem cells reveals characteristics of mature spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are adult stem cells in the testis of male animals and have the ability in self-renewal and differentiation. SSCs are derived from primordial germ cells (PGCs) that are mitotically arrested in the embryo before birth. Following the birth of the animal, PGCs resume mitosis and migrate from the centre of the seminiferous tubules to the basement membrane. The descendent of PGCs (also called gonocytes) establish stable SSC colonies in about a week postnatally in order to support the life-long spermatogenesis. Whether SSCs at different developmental stages differ in their molecular and cellular characteristics is currently unclear. In the presented study, we conducted bioinformatics analyses using transcriptomics data established previously in the laboratory on OCT4 (encoded by the pluripotent gene Pou5f1) expressing SSCs from the neonatal (3 days-post-partum, 3-dpp), juvenile (7-dpp) and adult (2~3-month) mice, including screen of differentially expressed genes (DEGs), protein-protein interaction (PPI) network analysis of DEGs and clustering of sub-networks from PPI. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were also performed on clustered sub-networks of the PPI. In addition, all genes were analyzed using GSEA (gene set enrichment analysis) based on GO, KEGG and HALLMARK gene sets. The results showed that SSCs have a large number of DEGs among OCT4-positive SSCs from neonatal, juvenile and adult mice. The distinguishable biological functions encoded by these DEGs include biosynthesis and energy metabolism, immune response, cell junction and expression of migration and cell differentiation-related genes. Significant changes in the cell membrane composition of OCT4-positive SSCs may not only cause hypersensitive immune reactions but also affect the cell-cell contact and responses to secreted cytokines in the extracellular environment. The results also suggest that OCT4-positive SSCs may shift metabolic state from oxidative phosphorylation to glycolysis and significantly reduce the transcription of genes related to ribosome formation during aging. These results provide new clues for future research on the regulatory mechanisms of male germline stem cell development, growth and aging.

Laboratory breeding of two Phortica species (Diptera: Drosophilidae), vectors of the zoonotic eyeworm Thelazia callipaeda

BackgroundSome species of drosophilid flies belonging to the genus Phortica feed on ocular secretions of mammals, acting as biological vectors of the zoonotic eyeworm Thelazia callipaeda. This study describes an effective breeding protocol of Phortica variegata and Phortica oldenbergi in insectary conditions.MethodsAlive gravid flies of P. oldenbergi, P. variegata and Phortica semivirgo were field collected in wooded areas of Lazio region (Italy) and allowed to oviposit singularly to obtain isofamilies. Flies were maintained in ovipots (200 ml) with a plaster-covered bottom to maintain high humidity level inside. Adult feeding was guaranteed by fresh apples and a liquid dietary supplement containing sodium chloride and mucin proteins, while larval development was obtained by Drosophila-like agar feeding medium. The breeding performances of two media were compared: a standard one based on cornmeal flour and an enriched medium based on chestnut flour. All conditions were kept in a climatic chamber with a photoperiod of 14:10 h light:dark, 26 ± 2 °C and 80 ± 10% RH.ResultsFrom a total of 130 field-collected Phortica spp., three generations (i.e. F1 = 783, F2 = 109, F3 = 6) were obtained. Phortica oldenbergi was the species with highest breeding performance, being the only species reaching F3. Chestnut-based feeding medium allowed higher adult production and survival probability in both P. oldenbergi and P. variegata. Adult production/female was promising in both species (P. oldenbergi: 13.5 F1/f; P. variegata: 4.5 F1/f).ConclusionsThis standardized breeding protocol, based on controlled climatic parameters and fly densities, together with the introduction of an enriched chestnut-based feeding medium, allowed to investigate aspects of life history traits of Phortica spp. involved in the transmission of T. callipaeda. Obtaining F3 generation of these species for the first time paved the road for the establishment of stable colonies, an essential requirement for future studies on these vectors in controlled conditions.Graphical abstract

Particle foraging strategies promote microbial diversity in marine environments.

Microbial foraging in patchy environments, where resources are fragmented into particles or pockets embedded in a large matrix, plays a key role in natural environments. In the oceans and freshwater systems, particle-associated bacteria can interact with particle surfaces in different ways: some colonize only during short transients, while others form long-lived, stable colonies. We do not yet understand the ecological mechanisms by which both short- and long-term colonizers can coexist. Here, we address this problem with a mathematical model that explains how marine populations with different detachment rates from particles can stably coexist. In our model, populations grow only while on particles, but also face the increased risk of mortality by predation and sinking. Key to coexistence is the idea that detachment from particles modulates both net growth and mortality, but in opposite directions, creating a trade-off between them. While slow-detaching populations show the highest growth return (i.e., produce more net offspring), they are more susceptible to suffer higher rates of mortality than fast-detaching populations. Surprisingly, fluctuating environments, manifesting as blooms of particles (favoring growth) and predators (favoring mortality) significantly expand the likelihood that populations with different detachment rates can coexist. Our study shows how the spatial ecology of microbes in the ocean can lead to a predictable diversification of foraging strategies and the coexistence of multiple taxa on a single growth-limiting resource.

Sympatric Populations of the Anopheles gambiae Complex in Southwest Burkina Faso Evolve Multiple Diverse Resistance Mechanisms in Response to Intense Selection Pressure with Pyrethroids.

Simple SummaryTargeting mosquitoes with insecticides is one of the most effective methods to prevent malaria transmission. Although numbers of malaria cases have declined substantially this century, this pattern is not universal and Burkina Faso has one of the highest burdens of malaria; it is also a hotspot for the evolution of insecticide resistance in malaria vectors. We have established laboratory colonies from multiple species within the An. gambiae complex, the most efficient group of malaria vectors in the world, from larval collections in southwest Burkina Faso. Using bioassays with different insecticides widely used to control public health pests, we provide a profile of insecticide resistance in each of these colonies and, using molecular tools, reveal the genetic changes underpinning this resistance. We show that, whilst many resistance mechanisms are shared between species, there are some important differences which may affect resistance to current and future insecticide classes. The complexity, and diversity of resistance mechanisms highlights the importance of screening any potential new insecticide intended for use in malaria control against a wide range of populations. These stable laboratory colonies provide a valuable resource for insecticide discovery, and for further studies on the evolution and dispersal of insecticide resistance within and between species.Pyrethroid resistance in the Anopheles vectors of malaria is driving an urgent search for new insecticides that can be used in proven vector control tools such as insecticide treated nets (ITNs). Screening for potential new insecticides requires access to stable colonies of the predominant vector species that contain the major pyrethroid resistance mechanisms circulating in wild populations. Southwest Burkina Faso is an apparent hotspot for the emergence of pyrethroid resistance in species of the Anopheles gambiae complex. We established stable colonies from larval collections across this region and characterised the resistance phenotype and underpinning genetic mechanisms. Three additional colonies were successfully established (1 An. coluzzii, 1 An. gambiae and 1 An. arabiensis) to add to the 2 An. coluzzii colonies already established from this region; all 5 strains are highly resistant to pyrethroids. Synergism assays found that piperonyl butoxide (PBO) exposure was unable to fully restore susceptibility although exposure to a commercial ITN containing PBO resulted in 100% mortality. All colonies contained resistant alleles of the voltage gated sodium channel but with differing proportions of alternative resistant haplotypes. RNAseq data confirmed the role of P450s, with CYP6P3 and CYP6Z2 elevated in all 5 strains, and identified many other resistance mechanisms, some found across strains, others unique to a particular species. These strains represent an important resource for insecticide discovery and provide further insights into the complex genetic changes driving pyrethroid resistance.

Use of directed enzyme evolution to create novel biosynthetic pathways for production of rare or non-natural carotenoids.

In recent years, advances in bioengineering and synthetic biology techniques have been used to create carotenoid diversity in the laboratory. In this chapter, we describe the step-by-step method to perform directed evolution of carotenoid biosynthetic enzymes. We first explain how to establish an efficient Escherichia coli colony-based screening, including a detailed description of plasmid DNA construction design as well as tips and tricks to handle and manipulate cells to produce stable colonies. As an example for the directed evolution experiment, we engineer a bacterial phytoene desaturase CrtI to obtain a C50-phytoene desaturase, which catalyzes formation of a non-natural long-chain carotenoid. The method described in this chapter can be applied to many carotenoid biosynthetic enzymes, whose numbers have been rapidly expanding with recent advances in genomics. The use of directed evolution for carotenoid enzymes will contribute not only to the discovery of novel carotenoids but also to a deeper understanding of the creation and evolution of carotenoid biosynthetic pathways in nature.

Establishing and Maintaining an Etruscan Shrew Colony.

The Etruscan shrew (Suncus etruscus) is one of the smallest mammals on earth and is used in many fields of research, including physiology, behavioral science and neuroscience. However, establishing and maintaining a breeding colony of this species in the laboratory can be challenging, as it requires specific husbandry conditions that greatly differ from those of more common laboratory species such as mice or rats. Over the past 15 y, we have successfully established a long-term thriving colony of 150 to 200 animals originating from 36 founders. The colony shows longer life expectancy and larger litter sizes than wild conspecifics. Breeding occurs year-round, independent of seasons, and a breeding pair can regularly produce 2 to 6 offspring with an average life expectancy of more than 3 y. The shrews are housed in glass or plastic enclosures on a specific soil-sand-mixture bedding and are provided with hideouts and nesting material consisting of moss, wood, or bark. Due to their high basal metabolic rate, the shrews require food intake greater than their body weight per day, can hunt arthropods as large as themselves, and cannot survive more than a few hours without food. Live feed such as crickets or mealworms is crucial and must be provided daily or, at the very least, every 2 d. Although our husbandry practices have constantly been adapted and refined, shrew husbandry remains challenging, and great care is necessary to meet the specific needs of this species. Here, we describe the establishment of a long-term stable colony of Etruscan shrews in a research animal facility and the specific husbandry requirements for animal wellbeing.

Candida intermedia CBS 141442: A Novel Glucose/Xylose Co-Fermenting Isolate for Lignocellulosic Bioethanol Production

The present study describes the isolation of the novel strain Candida intermedia CBS 141442 and investigates the potential of this microorganism for the conversion of lignocellulosic streams. Different C. intermedia clones were isolated during an adaptive laboratory evolution experiment under the selection pressure of lignocellulosic hydrolysate and in strong competition with industrial, xylose-fermenting Saccharomyces cerevisiae cells. Isolates showed different but stable colony and cell morphologies when growing in a solid agar medium (smooth, intermediate and complex morphology) and liquid medium (unicellular, aggregates and pseudohyphal morphology). Clones of the same morphology showed similar fermentation patterns, and the C. intermedia clone I5 (CBS 141442) was selected for further testing due to its superior capacity for xylose consumption (90% of the initial xylose concentration within 72 h) and the highest ethanol yields (0.25 ± 0.02 g ethanol/g sugars consumed). Compared to the well-known yeast Scheffersomyces stipitis, the selected strain showed slightly higher tolerance to the lignocellulosic-derived inhibitors when fermenting a wheat straw hydrolysate. Furthermore, its higher glucose consumption rates (compared to S. stipitis) and its capacity for glucose and xylose co-fermentation makes C. intermedia CBS 141442 an attractive microorganism for the conversion of lignocellulosic substrates, as demonstrated in simultaneous saccharification and fermentation processes.

Generation and initial characterization of a collection of spontaneous Streptomyces albus J1074 mutants resistant to rifampicin

Aim. Streptomyces albus J1074 is one of the most popular streptomycete chassis for heterologous expression of natural product (NP) biosynthetic gene clusters (BGCs). There is keen interest in further improvement of the strain to provide increased yields of corresponding NPs. Introduction of certain types of antibiotic resistance mutations is a proven way to improve Streptomyces strains. For example, selection for increased resistance to rifampicin is known to lead to increased antibiotic activity. Here we used available lineages of antibiotic-resistant mutants of S. albus to raise rifampicin-resistant variants (Rifr) and to study their properties. Methods. Microbiological and molecular genetic approaches were combined to generate Rifr mutants and to study their properties. Results. By plating S. albus onto GYM agar supplemented with 10 mcg/mL of rifampicin, we isolated 85 stable Rifr colonies, whose resistance level was within 10-200 mcg/mL range. Sequencing revealed wide spectrum of missense mutations within rpoB gene. Bioassays demonstrated dramatically increased endogenous antibiotic activity of certain Rifr mutants. Conclusions. Selection for rifampicin resistance is a viable way to increase the yields of NPs in S. albus.
 Keywords: Streptomyces albus J1074, antibiotic resistance, rifampicin.

Crary bank: a deep foraging habitat for emperor penguins in the western Ross Sea

Although most dives of emperor penguins (Aptenodytes forsteri) are less than 100 m, penguins from the Cape Washington colony regularly perform deep dives > 400 m. To evaluate the significance and location of these deep dives of birds on foraging trips from Cape Washington, we report the satellite tracks of three birds. We also review the frequency of deep dives in the 35 of 42 birds that performed deep dives during seven research seasons over 22 years. Records included 83,314 dives, of which 1418 were > 400 m deep (deepest 552 m). Durations of these deep dives ranged from 7 to 13 min, up to more than twice the aerobic dive limit. Inter-deep-dive-intervals (IDDIs) between most deep dives were 10–20 min. The travel routes of satellite-tagged birds showed that all three spent time over Crary Bank, about 100 km from Cape Washington. Dives > 400 m only occurred over Crary Bank in the two satellite-tracked birds that were also equipped with dive recorders. The depths of the dives were consistent with the distribution of the most common, and energy-dense prey item found in their diet, Pleuragramma antarctica. We conclude that significant food resources are located over Crary Bank, accounting for the deep dives and success of birds from Cape Washington, the second largest, stable colony of emperor penguins known.

An aphid RNA transcript migrates systemically within plants and is a virulence factor

Aphids are sap-feeding insects that colonize a broad range of plant species and often cause feeding damage and transmit plant pathogens, including bacteria, viruses, and viroids. These insects feed from the plant vascular tissue, predominantly the phloem. However, it remains largely unknown how aphids, and other sap-feeding insects, establish intimate long-term interactions with plants. To identify aphid virulence factors, we took advantage of the ability of the green peach aphid Myzus persicae to colonize divergent plant species. We found that a M. persicae clone of near-identical females established stable colonies on nine plant species of five representative plant eudicot and monocot families that span the angiosperm phylogeny. Members of the novel aphid gene family Ya are differentially expressed in aphids on the nine plant species and are coregulated and organized as tandem repeats in aphid genomes. Aphids translocate Ya transcripts into plants, and some transcripts migrate to distal leaves within several plant species. RNAi-mediated knockdown of Ya genes reduces M. persicae fecundity, and M. persicae produces more progeny on transgenic plants that heterologously produce one of the systemically migrating Ya transcripts as a long noncoding (lnc) RNA. Taken together, our findings show that beyond a range of pathogens, M. persicae aphids translocate their own transcripts into plants, including a Ya lncRNA that migrates to distal locations within plants, promotes aphid fecundity, and is a member of a previously undescribed host-responsive aphid gene family that operate as virulence factors.

Antimicrobial and antioxidant characteristics of exopolysaccharides produced by Agrobacterium tumefaciens Cs5 and T1

This work was conducted to isolate and screen for bacteria that producing higher quantities of exopolysaccharides (EPSs) from the Egyptian soils. Forty soil samples were collected from different regions of three Governorates (Beheira, Giza, and Qalyubia). A total of 165 bacterial isolates were obtained from the soil rhizosphere. Thirty-five of them (21.2%) had ability to produce stable ropy colonies. After screening, two isolates Cs5 and T1 were selected for further study according to their capacity to produce a high quantity of EPS (7.1 and 5.7 g/l, respectively). The selected bacterial isolates were identified as non-similar strains of Agrobacterium tumefaciens based on their morphological, biochemical characteristics as well as 16S rRNA gene sequencing. To obtain maximum EPS production of both strains, nutritional and cultivation parameters were optimized using the one-factor-at-a-time method. Thus, the maximum EPS yield of 21.63 and 19.57 g/l were achieved from the strains Cs5 and T1, respectively. When molasses and corn steep liquor were substituted the synthetic carbon and nitrogen sources as low cost-substrates, EPS yields were improved to be 28.73 and 24.5 g/l for Cs5 and T1, respectively. The infrared (FT-IR) spectra for both extracts revealed the typical patterns of polysaccharide absorption. Moreover, HPLC analysis demonstrated that EPSs were heteropolysaccharide composed of glucosamine, glucose, and fructose. The produced EPS showed antioxidant capacity as well as strong antibacterial activity against Staphylococcus aureus (methicillin-resistant) and Bacillus cereus as hazardous human pathogens, suggesting their application in the food industry. While the produced EPS did not affect cancer cells.