NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization‐defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I‐E CRISPR‐associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild‐type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.     

The southernmost worm, <Emphasis Type="Italic">Scottnema lindsayae</Emphasis> (Nematoda): diversity,dispersal and ecological stability

The nematode worm Scottnema lindsayae (Cephalobidae) was found near the base of the Beardmore Glacier in the Transantarctic Mountains 83.48°S, over 5° further south than previously recorded. Identification was confirmed using morphological analyses of males, females and juvenile stages, and by DNA sequencing of the ITS1 region of the ribosomal RNA tandem repeat unit. These data revealed no discernable morphological or ITS rDNA sequence variation between the extreme southern population of S. lindsayae and disparate populations from the McMurdo Dry Valleys in south Victoria Land (77–78°S). Based on these results, we suggest that broadcast dispersal, with accompanying high rates of gene flow, establish the extreme southern distribution of the phylum Nematoda. High abundance, low rates of diversification and lack of an apparent biogeographic structure across latitudinal and environmental gradients implies that their presence in simple Antarctic soil ecosystems is stable, so long as physical and biological controls on their distributions remain within viable parameters. Recent evidence that S. lindsayae populations are in decline suggests that their high dispersal rates are insufficient to buffer current, unfavorable environmental changes and may foreshadow longer-term ecosystem disruption.     

Historical trends in frog populations in New Zealand based on public perceptions

Surveys were distributed to New Zealand land users in 1998 and 2008 to acquire information about New Zealand frogs with the aim of compiling and mapping their distribution and inferred population trends without costly and time-consuming field surveys. The overall frog population trend was reported as declining, with possible causes reported as an increase in agriculture, an increase in the distribution of predatory fish and disease. The resultant maps could be used for four main purposes: 1) to identify regions where Litoria populations are known to occur, which can be eliminated when considering suitable regions for translocation of Leiopelma; 2) to identify growing or stable populations of Litoria species, which may assist future disease surveys, population monitoring and to identify sources of genetic material that may serve as an Ark for declining Australian populations; 3) to highlight populations that are in decline to enable effective targeting of detailed disease studies; and 4) to approximate the stability of amphibian populations in the absence of more accurate, but costly, scientific monitoring.     

A novel harmony search-K means hybrid algorithm for clustering gene expression data

Recent progress in bioinformatics research has led to the accumulation of huge quantities of biological data at various data sources. The DNA microarray technology makes it possible to simultaneously analyze large number of genes across different samples. Clustering of microarray data can reveal the hidden gene expression patterns from large quantities of expression data that in turn offers tremendous possibilities in functional genomics, comparative genomics, disease diagnosis and drug development. The k- ¬means clustering algorithm is widely used for many practical applications. But the original k-¬means algorithm has several drawbacks. It is computationally expensive and generates locally optimal solutions based on the random choice of the initial centroids. Several methods have been proposed in the literature for improving the performance of the k-¬means algorithm. A meta-heuristic optimization algorithm named harmony search helps find out near-global optimal solutions by searching the entire solution space. Low clustering accuracy of the existing algorithms limits their use in many crucial applications of life sciences. In this paper we propose a novel Harmony Search-K means Hybrid (HSKH) algorithm for clustering the gene expression data. Experimental results show that the proposed algorithm produces clusters with better accuracy in comparison with the existing algorithms.     

Previously unrecognized stages of species‐specific colonization in the mutualism between Xenorhabdus bacteria and Steinernema nematodes

The specificity of a horizontally transmitted microbial symbiosis is often defined by molecular communication between host and microbe during initial engagement, which can occur in discrete stages. In the symbiosis between Steinernema nematodes and Xenorhabdus bacteria, previous investigations focused on bacterial colonization of the intestinal lumen (receptacle) of the nematode infective juvenile (IJ), as this was the only known persistent, intimate and species‐specific contact between the two. Here we show that bacteria colonize the anterior intestinal cells of other nematode developmental stages in a species‐specific manner. Also, we describe three processes that only occur in juveniles that are destined to become IJs. First, a few bacterial cells colonize the nematode pharyngeal‐intestinal valve (PIV) anterior to the intestinal epithelium. Second, the nematode intestine constricts while bacteria initially remain in the PIV. Third, anterior intestinal constriction relaxes and colonizing bacteria occupy the receptacle. At each stage, colonization requires X. nematophila symbiosis region 1 (SR1) genes and is species‐specific: X. szentirmaii, which naturally lacks SR1, does not colonize unless SR1 is ectopically expressed. These findings reveal new aspects of Xenorhabdus bacteria interactions with and transmission by theirSteinernema nematode hosts, and demonstrate that bacterial SR1 genes aid in colonizing nematode epithelial surfaces.     

Outcrossing and crossbreeding recovers deteriorated traits in laboratory cultured Steinernema carpocapsae nematodes

The nematode Steinernema carpocapsae infects and kills many pest insects in agro-ecosystems and is commonly used in biocontrol of these pests. Growth of the nematodes prior to distribution for biocontrol commonly results in deterioration of traits that are essential for nematode persistence in field applications. To better understand the mechanisms underlying trait deterioration of the efficacy of natural parasitism in entomopathogenic nematodes, we explored the maintenance of fitness related traits including reproductive capacity, heat tolerance, virulence to insects and ‘tail standing’ (formerly called nictation) among laboratory-cultured lines derived from natural, randomly mating populations of S. carpocapsae. Laboratory cultured nematode lines with fitness-related trait values below wild-type levels regained wild-type levels of reproductive and heat tolerance traits when outcrossed with a non-deteriorated line, while virulence and ‘tail standing’ did not deteriorate in our experiments. Crossbreeding two trait-deteriorated lines with each other also resulted in restoration of trait means to wild-type levels in most crossbred lines. Our results implicate inbreeding depression as the primary cause of trait deterioration in the laboratory cultured S. carpocapsae. We further suggest the possibility of creating inbred lines purged of deleterious alleles as founders in commercial nematode growth.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.     

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic ^1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis ⁴. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects ⁵. For transmission between insect hosts, the bacteria colonize the intestine of the nematode''s infective juvenile stage ^6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects ^9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems ⁹.We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila¹⁴. Similar methods have been used to investigate other nematode-bacterium associations ^9,15-18and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization ^14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues ^14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication ²²or grinding ²³, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes ^21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization ^17,18, and is less laborious than other methods, including sonication ^22,25-27and individual nematode dissection ^28,29.