Paraburkholderia largidicola sp. nov., a gut symbiont of the bordered plant bug Physopelta gutta.

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated strains F2T and PGU16, were isolated from the midgut crypts of the bordered plant bug Physopelta gutta, collected in Okinawa prefecture, Japan. Although these strains were derived from different host individuals collected at different times, their 16S rRNA gene sequences were identical and showed the highest similarity to Paraburkholderia caribensis MWAP64T (99.3 %). The genome of strain F2T consisted of two chromosomes and two plasmids, and its size and G+C content were 9.28 Mb and 62.4 mol% respectively; on the other hand, that of strain PGU16 consisted of two chromosomes and three plasmids, and its size and G+C content were 9.47 Mb and 62.4 mol%, respectively. Phylogenetic analyses revealed that these two strains are members of the genus Paraburkholderia. The digital DNA-DNA hybridization value between these two strains was 92.4 %; on the other hand, the values between strain F2T and P. caribensis MWAP64T or phylogenetically closely related Paraburkholderia species were 44.3 % or below 49.1 %. The predominant fatty acids of both strains were C16 : 0, C17 : 0 cyclo, summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), and C19 : 0 cyclo ω8c, and their respiratory quinone was ubiquinone 8. Based on the above genotypic and phenotypic characteristics, strains F2T and PGU16 represent a novel species of the genus Paraburkholderia for which the name Paraburkholderia largidicola sp. nov. is proposed. The type strain is F2T (=NBRC 115765T=LMG 32765T).

High Prevalence of Pantoea spp. in Microbiota Associated with the Sorghum Plant Bug Stenotus rubrovittatus (Heteroptera: Miridae).

The sorghum plant bug, Stenotus rubrovittatus (order Heteroptera: family Miridae), is a notorious insect pest in Japan that causes pecky rice. In the present study, we sampled this insect pest in the northern part of Honshu Island in Japan and investigated its associated microbiota. The results obtained showed that Pantoea dominated the associated microbiota and was the sole genus detected in all samples. The dominant Pantoea were phylogenetically close to rice pathogens. The present results suggest that the sorghum plant bug needs to be regarded and controlled not only as a notorious insect pest, but also as a potential vector of rice pathogenic Pantoea spp.

Intercontinental Diversity of Caballeronia Gut Symbionts in the Conifer Pest Bug Leptoglossus occidentalis.

Many stinkbugs in the superfamily Coreoidea (Hemiptera: Heteroptera) develop crypts in the posterior midgut, harboring Caballeronia (Burkholderia) symbionts. These symbionts form a monophyletic group in Burkholderia sensu lato, called the "stinkbug-associated beneficial and environmental (SBE)" group, recently reclassified as the new genus Caballeronia. SBE symbionts are separated into the subclades SBE-α and SBE-ß. Previous studies suggested a regional effect on the symbiont infection pattern; Japanese and American bug species are more likely to be associated with SBE-α, while European bug species are almost exclusively associated with SBE-ß. However, since only a few insect species have been investigated, it remains unclear whether region-specific infection is general. We herein investigated Caballeronia gut symbionts in diverse Japanese, European, and North American populations of a cosmopolitan species, the Western conifer seed bug Leptoglossus occidentalis (Coreoidea: Coreidae). A mole-cular phylogenetic ana-lysis of the 16S rRNA gene demonstrated that SBE-ß was the most dominant in all populations. Notably, SBE-α was rarely detected in any region, while a third clade, the "Coreoidea clade" occupied one fourth of the tested populations. Although aposymbiotic bugs showed high mortality, SBE-α- and SBE-ß-inoculated insects both showed high survival rates; however, a competition assay demonstrated that SBE-ß outcompeted SBE-α in the midgut crypts of L. occidentalis. These results strongly suggest that symbiont specificity in the Leptoglossus-Caballeronia symbiotic association is influenced by the host rather than geography, while the geographic distribution of symbionts may be more important in other bugs.

Complete Genome Sequence of "Candidatus Hydrogeosomobacter endosymbioticus," an Intracellular Bacterial Symbiont of the Anaerobic Ciliate Scuticociliate GW7.

The bacterium "Candidatus Hydrogenosomobacter endosymbioticus" is an intracellular symbiont of anaerobic scuticociliate GW7, which is associated with hydrogenosome together with methanogenic archaea. Here, we report a complete genome sequence of the symbiont consisting of 827 kbp. Knowing this sequence would contribute to the understanding of the metabolic interactions and evolution of the tripartite symbiosis.

Illustrating a Species Sensitivity Distribution for Nano- and Microplastic Particles Using Bayesian Hierarchical Modeling.

Environmental contamination with nano- and microplastic (NMP) particles is an emerging global concern. The derivation of species sensitivity distributions (SSDs) is an essential step in estimating a hazardous concentration for 5% of the species (HC5), and this HC5 value is often used as a "safe" concentration in ecological risk assessment, that is, predicted-no-effect concentration. Although properties of plastics such as particle size can affect toxic effect concentrations, such influences have not yet been quantitatively considered in estimating SSDs for NMP particles. We illustrate a log-normal SSD using chronic lowest-observed-effect concentrations (LOECs) of NMP particles from readily available toxicity data sets, considering the influence of particle size, polymer type, and freshwater or marine test media by adopting Bayesian hierarchical modeling techniques. Results of the hierarchical SSD modeling suggest that the SSD mean was negatively associated with particle size and was lower in marine media than in freshwater media. The posterior medians of the HC5 estimated from the LOEC-based SSD varied by a factor of 10 depending on these factors (e.g., 1.8-20 µg/L for the particle size range of 0.1-5000 µm in the marine environment). Hierarchical SSD modeling allows us to clarify the influences of important factors such as NMP properties on effect concentrations, thereby helping to guide more relevant ecological risk assessments for NMP. Environ Toxicol Chem 2022;41:954-960. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

What do we want to estimate from observational datasets? Choosing appropriate statistical analysis methods based on the chemical management phase.

The goals of observational dataset analysis vary with the management phase of environments threatened by anthropogenic chemicals. For example, identifying severely compromised sites is necessary to determine candidate sites in which to implement measures during early management phases. Among the most effective approaches is developing regression models with high predictive power for dependent variable values using the Akaike information criterion. However, this analytical approach may be theoretically inappropriate to obtain the necessary information in various chemical management phases, such as the intervention effect size of a chemical required in the late chemical management phase to evaluate the necessity of an effluent standard and its specific value. However, choosing appropriate statistical methods based on the data analysis objective in each chemical management phase has rarely been performed. This study provides an overview of the primary data analysis objectives in the early and late chemical management phases. For each objective, several suitable statistical analysis methods for observational datasets are detailed. In addition, the study presents examples of linear regression analysis procedures using an available dataset derived from field surveys conducted in Japanese rivers. Integr Environ Assess Manag 2022;18:1414-1422. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Insecticide resistance by a host-symbiont reciprocal detoxification.

Insecticide resistance is one of the most serious problems in contemporary agriculture and public health. Although recent studies revealed that insect gut symbionts contribute to resistance, the symbiont-mediated detoxification process remains unclear. Here we report the in vivo detoxification process of an organophosphorus insecticide, fenitrothion, in the bean bug Riptortus pedestris. Using transcriptomics and reverse genetics, we reveal that gut symbiotic bacteria degrade this insecticide through a horizontally acquired insecticide-degrading enzyme into the non-insecticidal but bactericidal compound 3-methyl-4-nitrophenol, which is subsequently excreted by the host insect. This integrated "host-symbiont reciprocal detoxification relay" enables the simultaneous maintenance of symbiosis and efficient insecticide degradation. We also find that the symbiont-mediated detoxification process is analogous to the insect genome-encoded fenitrothion detoxification system present in other insects. Our findings highlight the capacity of symbiosis, combined with horizontal gene transfer in the environment, as a powerful strategy for an insect to instantly eliminate a toxic chemical compound, which could play a critical role in the human-pest arms race.

A Peptidoglycan Amidase Mutant of Burkholderia insecticola Adapts an L-form-like Shape in the Gut Symbiotic Organ of the Bean Bug Riptortus pedestris.

Bacterial cell shapes may be altered by the cell cycle, nutrient availability, environmental stress, and interactions with other organisms. The bean bug Riptortus pedestris possesses a symbiotic bacterium, Burkholderia insecticola, in its midgut crypts. This symbiont is a typical rod-shaped bacterium under in vitro culture conditions, but changes to a spherical shape inside the gut symbiotic organ of the host insect, suggesting the induction of morphological alterations in B. insecticola by host factors. The present study revealed that a deletion mutant of a peptidoglycan amidase gene (amiC), showing a filamentous chain form in vitro, adapted a swollen L-form-like cell shape in midgut crypts. Spatiotemporal observations of the ΔamiC mutant in midgut crypts revealed the induction of swollen cells, particularly prior to the molting of insects. To elucidate the mechanisms underlying in vivo-specific morphological alterations, the symbiont was cultured under 13 different conditions and its cell shape was examined. Swollen cells, similar to symbiont cells in midgut crypts, were induced when the mutant was treated with fosfomycin, an inhibitor of peptidoglycan precursor biosynthesis. Collectively, these results strongly suggest that the Burkholderia symbiont in midgut crypts is under the control of the host insect via a cell wall-attacking agent.

The effect of intervention in nickel concentrations on benthic macroinvertebrates: A case study of statistical causal inference in ecotoxicology.

Field survey-based ecological risk assessments for trace metals are conducted to examine the necessity and/or effectiveness of management intervention, such as setting of environmental quality standards. Observational datasets often involve confounders that may bias estimation of the effects of intervention (e.g., reduction of trace-metal concentrations through regulation). The field of ecotoxicology lags behind some other research fields in understanding proper analytical procedures for causal inference from observational datasets; there are only a few field survey-based ecotoxicological studies that have explicitly controlled for confounders in their statistical analyses. In the present study, we estimated the effect of intervention in nickel concentrations on Ephemeroptera, Plecoptera, and Trichoptera richness in rivers in Japan. We also provide detailed explanations for the backgrounds of spurious associations derived from confounders and on proper analytical procedures for obtaining an unbiased estimate of the targeted intervention effect by using regression analysis. We constructed a multiple regression model based on a causal diagram for aquatic insects and environmental factors, and on "the backdoor criterion," that enabled us to determine the set of covariates required to obtain an unbiased estimate of the targeted intervention effect from regression coefficients. We found that management intervention in nickel concentrations may be ineffective compared to intervention in organic pollution, and that analysis ignoring the confounders overestimated the effect of intervention in nickel concentrations. Our results highlight the fact that confounders can lead to misjudging the necessity for management of anthropogenic chemical substances. Confounders should be explicitly specified and statistically controlled to achieve a comprehensive assessment of ecological risks for various substances.

Evaluation of interregional consistency in associations between neonicotinoid insecticides and functions of benthic invertebrate communities in rivers in urban rice-paddy areas.

Neonicotinoid insecticides pose risks that need to be managed for conservation of aquatic ecosystems. In this study, we evaluated the associations between the estimated environmental concentrations of two neonicotinoid insecticides (imidacloprid and dinotefuran) and the total abundances of seven functional feeding groups (FFGs) of benthic invertebrate communities in rivers in urban rice-paddy areas in four Japanese regions. Regional datasets of benthic invertebrate communities and environmental variables available for Japan were analyzed. The associations between neonicotinoid exposure and benthic functional groups in each region were evaluated by applying a partial redundancy analysis to each regional dataset. We then examined whether there was an interregionally consistent pattern in the observed associations to ascertain the general applicability of the associations. In two of the four regions, the associations of the total abundances of the seven FFGs with neonicotinoid concentrations were significant, suggesting negative effects of imidacloprid and dinotefuran on river ecosystem functions in these two Japanese regions. Moreover, although the associations in the remaining two regions were not significant, the pattern of associations of the total abundances of six of the FFGs (shredders, filter feeders, collectors, grazers, predators, and scavengers) with imidacloprid concentrations seemed to be consistent among the four regions. This implies broad-scale negative effects of imidacloprid on river ecosystem functions in urban rice-paddy areas. We did not, however, find any interregionally consistent patterns in the associations with dinotefuran concentrations. This may be related to the multicollinearity with the imidacloprid concentrations and/or the low maximum dinotefuran concentration relative to the toxicity values of this neonicotinoid. Therefore, the association between dinotefuran and river ecosystem functions requires additional investigation. We believe that this type of hypotheses-generating research using country-wide biomonitoring and exposure databases can be a great aid in future ecological risk assessment studies.

Genomic Comparison of Insect Gut Symbionts from Divergent Burkholderia Subclades.

Stink bugs of the superfamilies Coreoidea and Lygaeoidea establish gut symbioses with environmentally acquired bacteria of the genus Burkholderia sensu lato. In the genus Burkholderia, the stink bug-associated strains form a monophyletic clade, named stink bug-associated beneficial and environmental (SBE) clade (or Caballeronia). Recently, we revealed that members of the family Largidae of the superfamily Pyrrhocoroidea are associated with Burkholderia but not specifically with the SBE Burkholderia; largid bugs harbor symbionts that belong to a clade of plant-associated group of Burkholderia, called plant-associated beneficial and environmental (PBE) clade (or Paraburkholderia). To understand the genomic features of Burkholderia symbionts of stink bugs, we isolated two symbiotic Burkholderia strains from a bordered plant bug Physopellta gutta (Pyrrhocoroidea: Largidae) and determined their complete genomes. The genome sizes of the insect-associated PBE (iPBE) are 9.5 Mb and 11.2 Mb, both of which are larger than the genomes of the SBE Burkholderia symbionts. A whole-genome comparison between two iPBE symbionts and three SBE symbionts highlighted that all previously reported symbiosis factors are shared and that 282 genes are specifically conserved in the five stink bug symbionts, over one-third of which have unknown function. Among the symbiont-specific genes, about 40 genes formed a cluster in all five symbionts; this suggests a "symbiotic island" in the genome of stink bug-associated Burkholderia.

Complete Genome Sequence of Burkholderia sp. Strain THE68, a Bacterial Symbiont Isolated from Midgut Crypts of the Seed Bug Togo hemipterus.

Burkholderia sp. strain THE68 is a bacterial symbiont isolated from the midgut crypts of a phytophagous stink bug, Togo hemipterus Here, we report the complete 7.98-Mb genome of this symbiont, which consists of six circular replicons containing 7,238 protein coding genes.

Host-symbiont specificity determined by microbe-microbe competition in an insect gut.

Despite the omnipresence of specific host-symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont.

Tripartite Symbiosis of an Anaerobic Scuticociliate with Two Hydrogenosome-Associated Endosymbionts, a Holospora-Related Alphaproteobacterium and a Methanogenic Archaeon.

A number of anaerobic ciliates, unicellular eukaryotes, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from a different domain, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has previously been described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria, which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we propose a novel candidate genus and species, "Candidatus Hydrogenosomobacter endosymbioticus."IMPORTANCE Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

Associations of Community Structure and Functions of Benthic Invertebrates with Nickel Concentrations: Analyses from Field Surveys.

Field surveys provide valuable empirical information about the effects of heavy metals on the biological integrity of river ecosystems. To evaluate the effect of nickel (Ni) on aquatic organisms, we conducted field surveys of benthic invertebrates and Ni concentrations at 45 sites in 19 rivers in Japan. We examined the relationships between 11 structural or functional measures of benthic invertebrate communities and free Ni ion concentrations with a 90th quantile regression model. Among the measures, Ephemeroptera, Plecoptera, and Trichoptera (EPT) richness, total wet biomass of all invertebrates, and total abundance of filter feeders were negatively associated with free Ni ion concentrations. The total abundance of mud burrowers and their percentage contribution to the abundance of all invertebrates as well as the total abundances of collector-gatherers and predators were positively associated with the Ni ion concentrations. The free ion concentrations of Ni associated with a 5% reduction of one of the 3 measures from its 90th quantile value at reference sites (EC5) were estimated to be 0.2 to 7.6 µg/L. The point estimates of EC5 were similar in order of magnitude to the environmental quality standard for Ni in the European Union. However, the usefulness of simple point estimates of effective concentrations based on quantile regression lines should be carefully examined because the uncertainties in our EC5 values were large. Environ Toxicol Chem 2019;38:1728-1737. © 2019 SETAC.

Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture.

In the symbiosis of the bean bug Riptortus pedestris with Burkholderia insecticola, the bacteria occupy an exclusive niche in the insect midgut and favor insect development and reproduction. In order to understand how the symbiotic bacteria stably colonize the midgut crypts and which services they provide to the host, we compared the cytology, physiology, and transcriptomics of free-living and midgut-colonizing B. insecticola. The analyses revealed that midgut-colonizing bacteria were smaller in size and had lower DNA content, they had increased stress sensitivity, lost motility, and an altered cell surface. Transcriptomics revealed what kinds of nutrients are provided by the bean bug to the Burkholderia symbiont. Transporters and metabolic pathways of diverse sugars such as rhamnose and ribose, and sulfur compounds like sulfate and taurine were upregulated in the midgut-colonizing symbionts. Moreover, pathways enabling the assimilation of insect nitrogen wastes, i.e. allantoin and urea, were also upregulated. The data further suggested that the midgut-colonizing symbionts produced all essential amino acids and B vitamins, some of which are scarce in the soybean food of the host insect. Together, these findings suggest that the Burkholderia symbiont is fed with specific nutrients and also recycles host metabolic wastes in the insect gut, and in return, the bacterial symbiont provides the host with essential nutrients limited in the insect food, contributing to the rapid growth and enhanced reproduction of the bean bug host.

Burkholderia insecticola sp. nov., a gut symbiotic bacterium of the bean bug Riptortus pedestris.

A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64T, was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei clade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314T (100 %), Burkholderia turbans LMG 29316T (99.52 %) and Burkholderia ptereochthonis LMG 29326T (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome blast Distance Phylogeny confirmed B. peredens LMG 29314T, B. ptereochthonis LMG 29326T and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA-DNA hybridization experiments unambiguously demonstrated that strain RPE64T represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C16 : 0, C18 : 1ω7c and C17 : 0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64T from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64T is classified as representing a novel species of the genus Burkholderia, for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64T (=NCIMB 15023T=JCM 31142T).

Riptortus pedestris and Burkholderia symbiont: an ideal model system for insect-microbe symbiotic associations.

A number of insects establish symbiotic associations with beneficial microorganisms in various manners. The bean bug Riptortus pedestris and allied stink bugs possess an environmentally acquired Burkholderia symbiont in their midgut crypts. Unlike other insect endosymbionts, the Burkholderia symbiont is easily culturable and genetically manipulatable outside the host. In conjunction with the experimental advantages of the host insect, the Riptortus-Burkholderia symbiosis is an ideal model system for elucidating the molecular bases underpinning insect-microbe symbioses, which opens a new window in the research field of insect symbiosis. This review summarizes current knowledge of this system and discusses future perspectives.

Comparison of Drive Counts and Mark-Resight As Methods of Population Size Estimation of Highly Dense Sika Deer (Cervus nippon) Populations.

Assessing temporal changes in abundance indices is an important issue in the management of large herbivore populations. The drive counts method has been frequently used as a deer abundance index in mountainous regions. However, despite an inherent risk for observation errors in drive counts, which increase with deer density, evaluations of the utility of drive counts at a high deer density remain scarce. We compared the drive counts and mark-resight (MR) methods in the evaluation of a highly dense sika deer population (MR estimates ranged between 11 and 53 individuals/km2) on Nakanoshima Island, Hokkaido, Japan, between 1999 and 2006. This deer population experienced two large reductions in density; approximately 200 animals in total were taken from the population through a large-scale population removal and a separate winter mass mortality event. Although the drive counts tracked temporal changes in deer abundance on the island, they overestimated the counts for all years in comparison to the MR method. Increased overestimation in drive count estimates after the winter mass mortality event may be due to a double count derived from increased deer movement and recovery of body condition secondary to the mitigation of density-dependent food limitations. Drive counts are unreliable because they are affected by unfavorable factors such as bad weather, and they are cost-prohibitive to repeat, which precludes the calculation of confidence intervals. Therefore, the use of drive counts to infer the deer abundance needs to be reconsidered.

Comparative genomic analysis of retrogene repertoire in two green algae Volvox carteri and Chlamydomonas reinhardtii.

BACKGROUND: Retroposition, one of the processes of copying the genetic material, is an important RNA-mediated mechanism leading to the emergence of new genes. Because the transcription controlling segments are usually not copied to the new location in this mechanism, the duplicated gene copies (retrocopies) become pseudogenized. However, few can still survive, e.g. by recruiting novel regulatory elements from the region of insertion. Subsequently, these duplicated genes can contribute to the formation of lineage-specific traits and phenotypic diversity. Despite the numerous studies of the functional retrocopies (retrogenes) in animals and plants, very little is known about their presence in green algae, including morphologically diverse species. The current availability of the genomes of both uni- and multicellular algae provides a good opportunity to conduct a genome-wide investigation in order to fill the knowledge gap in retroposition phenomenon in this lineage. RESULTS: Here we present a comparative genomic analysis of uni- and multicellular algae, Chlamydomonas reinhardtii and Volvox carteri, respectively, to explore their retrogene complements. By adopting a computational approach, we identified 141 retrogene candidates in total in both genomes, with their fraction being significantly higher in the multicellular Volvox. Majority of the retrogene candidates showed signatures of functional constraints, thus indicating their functionality. Detailed analyses of the identified retrogene candidates, their parental genes, and homologs of both, revealed that most of the retrogene candidates were derived from ancient retroposition events in the common ancestor of the two algae and that the parental genes were subsequently lost from the respective lineages, making many retrogenes 'orphan'. CONCLUSION: We revealed that the genomes of the green algae have maintained many possibly functional retrogenes in spite of experiencing various molecular evolutionary events during a long evolutionary time after the retroposition events. Our first report about the retrogene set in the green algae provides a good foundation for any future investigation of the repertoire of retrogenes and facilitates the assessment of the evolutionary impact of retroposition on diverse morphological traits in this lineage. REVIEWERS: This article was reviewed by William Martin and Piotr Zielenkiewicz.