Secondary Endosymbionts Research Articles

Large-scale geography survey provides insights into the colonization history of a major aphid pest on its cultivated apple host in Europe, North America and North Africa

With frequent host shifts involving the colonization of new hosts across large geographical ranges, crop pests are good models for examining the mechanisms of rapid colonization. The microbial partners of pest insects may be involved or affected by colonization, which has been little studied so far. We investigated the demographic history of the rosy apple aphid, Dysaphis plantaginea, a major pest of the cultivated apple (Malus domestica) in Europe, North Africa and North America, as well as the diversity of its endosymbiotic bacterial community. We genotyped a comprehensive sample of 714 colonies from Europe, Morocco and the US using mitochondrial (CytB and CO1), bacterial (16s rRNA and TrnpB), and 30 microsatellite markers. We detected five populations spread across the US, Morocco, Western and Eastern Europe, and Spain. Populations showed weak genetic differentiation and high genetic diversity, except the Moroccan and the North American that are likely the result of recent colonization events. Coalescent-based inferences releaved high levels of gene flow among populations during the colonization, but did not allow determining the sequence of colonization of Europe, America and Morroco by D. plantaginea, likely because of the weak genetic differentiation and the occurrence of gene flow among populations. Finally, we found that D. plantaginea rarely hosts any other endosymbiotic bacteria than its obligate nutritional symbiont Buchnera aphidicola. This suggests that secondary endosymbionts did not play any role in the rapid spread of the rosy apple aphid. These findings have fundamental importance for understanding pest colonization processes and implications for sustainable pest control programs.

Bacillus subtilis (Bacillales, Bacillaceae) Spores Affect Survival and Population Growth in the Grain Aphid Sitobion avenae (Hemiptera, Aphididae) in Relation to the Presence of the Facultative Bacterial Endosymbiont Regiella insecticola (Enterobacteriales, Enterobacteriaceae).

The grain aphid Sitobion avenae (Fabricius) is one of the most important cereal pests, damaging crops through sap sucking and virus transmission. Sitobion avenae harbors the secondary endosymbiont Regiella insecticola, which is highly prevalent in populations in south-central Chile and other regions of the world. In order to develop ecological alternatives for biological control, we studied the effect of applying the spores of a strain of the bacterium Bacillus subtilis on the survival and fecundity of the most prevalent genotype of S. avenae in central Chile. The strain selected was one that in previous studies had shown the ability to outcompete other bacteria. Using clones of this aphid genotype infected and uninfected with R. insecticola, we found that applying B. subtilis spores through artificial diets and spraying on leaves decreased both adult survival and nymph production. The detection of spores within the aphid body was negatively correlated with nymph production and was lower in the presence of R. insecticola when applied in diets. B. subtilis spores applied on leaves reduced the number of aphids, an effect that was stronger on aphids harboring R. insecticola. A possible interaction between endosymbiotic bacteria and bacterial antagonists within the aphid body is discussed.

Horizontal Transfer of Microbial Toxin Genes to Gall Midge Genomes.

A growing body of evidence has underscored the role of horizontal gene transfer (HGT) in animal evolution. Previously, we discovered the horizontal transfer of the gene encoding the eukaryotic genotoxin cytolethal distending toxin B (cdtB) from the pea aphid Acyrthosiphon pisum secondary endosymbiont (APSE) phages to drosophilid and aphid nuclear genomes. Here, we report cdtB in the nuclear genome of the gall-forming “swede midge” Contarinia nasturtii (Diptera: Cecidomyiidae) via HGT. We searched all available gall midge genome sequences for evidence of APSE-to-insect HGT events and found five toxin genes (aip56, cdtB, lysozyme, rhs, and sltxB) transferred horizontally to cecidomyiid nuclear genomes. Surprisingly, phylogenetic analyses of HGT candidates indicated APSE phages were often not the ancestral donor lineage of the toxin gene to cecidomyiids. We used a phylogenetic signal statistic to test a transfer-by-proximity hypothesis for animal HGT, which suggested that microbe-to-insect HGT was more likely between taxa that share environments than those from different environments. Many of the toxins we found in midge genomes target eukaryotic cells, and catalytic residues important for toxin function are conserved in insect copies. This class of horizontally transferred, eukaryotic cell-targeting genes is potentially important in insect adaptation.

Co-Occurrence of Viruses, Plant Pathogens, and Symbionts in an Underexplored Hemipteran Clade.

Interactions between insect symbionts and plant pathogens are dynamic and complex, sometimes involving direct antagonism or synergy and sometimes involving ecological and evolutionary leaps, as insect symbionts transmit through plant tissues or plant pathogens transition to become insect symbionts. Hemipterans such as aphids, whiteflies, psyllids, leafhoppers, and planthoppers are well-studied plant pests that host diverse symbionts and vector plant pathogens. The related hemipteran treehoppers (family Membracidae) are less well-studied but offer a potentially new and diverse array of symbionts and plant pathogenic interactions through their distinct woody plant hosts and ecological interactions with diverse tending hymenopteran taxa. To explore membracid symbiont–pathogen diversity and co-occurrence, this study performed shotgun metagenomic sequencing on 20 samples (16 species) of treehopper, and characterized putative symbionts and pathogens using a combination of rapid blast database searches and phylogenetic analysis of assembled scaffolds and correlation analysis. Among the 8.7 billion base pairs of scaffolds assembled were matches to 9 potential plant pathogens, 12 potential primary and secondary insect endosymbionts, numerous bacteriophages, and other viruses, entomopathogens, and fungi. Notable discoveries include a divergent Brenneria plant pathogen-like organism, several bee-like Bombella and Asaia strains, novel strains of Arsenophonus-like and Sodalis-like symbionts, Ralstonia sp. and Ralstonia-type phages, Serratia sp., and APSE-type phages and bracoviruses. There were several short Phytoplasma and Spiroplasma matches, but there was no indication of plant viruses in these data. Clusters of positively correlated microbes such as yeast-like symbionts and Ralstonia, viruses and Serratia, and APSE phage with parasitoid-type bracoviruses suggest directions for future analyses. Together, results indicate membracids offer a rich palette for future study of symbiont–plant pathogen interactions.

Triple RNA-Seq characterizes aphid gene expression in response to infection with unequally virulent strains of the endosymbiont Hamiltonella defensa

BackgroundSecondary endosymbionts of aphids provide benefits to their hosts, but also impose costs such as reduced lifespan and reproductive output. The aphid Aphis fabae is host to different strains of the secondary endosymbiont Hamiltonella defensa, which encode different putative toxins. These strains have very different phenotypes: They reach different densities in the host, and the costs and benefits (protection against parasitoid wasps) they confer to the host vary strongly.ResultsWe used RNA-Seq to generate hypotheses on why four of these strains inflict such different costs to A. fabae. We found different H. defensa strains to cause strain-specific changes in aphid gene expression, but little effect of H. defensa on gene expression of the primary endosymbiont, Buchnera aphidicola. The highly costly and over-replicating H. defensa strain H85 was associated with strongly reduced aphid expression of hemocytin, a marker of hemocytes in Drosophila. The closely related strain H15 was associated with downregulation of ubiquitin-related modifier 1, which is related to nutrient-sensing and oxidative stress in other organisms. Strain H402 was associated with strong differential regulation of a set of hypothetical proteins, the majority of which were only differentially regulated in presence of H402.ConclusionsOverall, our results suggest that costs of different strains of H. defensa are likely caused by different mechanisms, and that these costs are imposed by interacting with the host rather than the host’s obligatory endosymbiont B. aphidicola.

Bemisia tabaci in Java, Indonesia: genetic diversity and the relationship with secondary endosymbiotic bacteria

Bemisia tabaci is a complex of cryptic species of whitefly distributed worldwide; they are serious agricultural pests and vectors of plant viruses. Whiteflies are commonly infected by endosymbiotic bacteria, but the infection profiles among genetic groups of B. tabaci are highly complex. Here we analyzed the genetic variation of B. tabaci and endosymbiont infection patterns in Java, Indonesia. Specifically, adult B. tabaci were collected from four provinces and 43 partial cytochrome oxidase subunit I gene sequences were determined to identify the genotypes. Results showed that B. tabaci was grouped into three different cryptic species, Asia I, Asia II 5, and Asia II 7, at rates of 90.70%, 6.98%, and 2.32%, respectively. The dominant group, Asia I, was distributed throughout the island, whereas Asia II 5 and Asia II 7 were detected only in West Java. In these cryptic species, the infection rates of the secondary endosymbionts Arsenophonus, Cardinium, Hamiltonella, Rickettsia, and Wolbachia were 37.21%, 72.09%, 37.21%, 88.37%, and 90.70%, respectively. Arsenophonus and Cardinium were detected two subgroups (A1 and A2; C2 and C4), but Hamiltonella, Rickettsia, and Wolbachia were detected a one subgroup (H1, R1, and W1). The A1 and A2 subgroups were distributed in a mixed manner across the entire island; however, the C2 and C4 subgroups were distributed differentially in West Java and in Central and East Java, respectively. Multiple infections were common and their patterns were highly variable in each cryptic species. In particular, Hamiltonella was detected in Asia I and Asia II 5 but never coinfected with Arsenophonus in the same individual. Overall, this study shows that Asia I is the dominant genetic B. tabaci group on Java Island and that infection by endosymbionts occurs in a highly complex and sometimes geographically related manner.

Genetic and endosymbiotic diversity of Greek populations of Philaenus spumarius, Philaenus signatus and Neophilaenus campestris, vectors of Xylella fastidiosa

The plant-pathogenic bacterium Xylella fastidiosa which causes significant diseases to various plant species worldwide, is exclusively transmitted by xylem sap-feeding insects. Given the fact that X. fastidiosa poses a serious potential threat for olive cultivation in Greece, the main aim of this study was to investigate the genetic variation of Greek populations of three spittlebug species (Philaenus spumarius, P. signatus and Neophilaenus campestris), by examining the molecular markers Cytochrome Oxidase I, cytochrome b and Internal Transcribed Spacer. Moreover, the infection status of the secondary endosymbionts Wolbachia, Arsenophonus, Hamiltonella, Cardinium and Rickettsia, among these populations, was determined. According to the results, the ITS2 region was the less polymorphic, while the analyzed fragments of COI and cytb genes, displayed high genetic diversity. The phylogenetic analysis placed the Greek populations of P. spumarius into the previously obtained Southwest clade in Europe. The analysis of the bacterial diversity revealed a diverse infection status. Rickettsia was the most predominant endosymbiont while Cardinium was totally absent from all examined populations. Philaenus spumarius harbored Rickettsia, Arsenophonus, Hamiltonella and Wolbachia, N. campestris carried Rickettsia, Hamiltonella and Wolbachia while P. signatus was infected only by Rickettsia. The results of this study will provide an important knowledge resource for understanding the population dynamics of vectors of X. fastidiosa with a view to formulate effective management strategies towards the bacterium.

Horizontal Transmission of the Heritable Protective Endosymbiont Hamiltonella defensa Depends on Titre and Haplotype.

Secondary endosymbionts of aphids have an important ecological and evolutionary impact on their host, as they provide resistance to natural enemies but also reduce the host’s lifespan and reproduction. While secondary symbionts of aphids are faithfully transmitted from mother to offspring, they also have some capacity to be transmitted horizontally between aphids. Here we explore whether 11 isolates from 3 haplotypes of the secondary endosymbiont Hamiltonella defensa differ in their capacity for horizontal transmission. These isolates vary in the protection they provide against parasitoid wasps as well as the costs they inflict on their host, Aphis fabae. We simulated natural horizontal transmission through parasitoid wasps by stabbing aphids with a thin needle and assessed horizontal transmission success of the isolates from one shared donor clone into three different recipient clones. Specifically, we asked whether potentially costly isolates reaching high cell densities in aphid hosts are more readily transmitted through this route. This hypothesis was only partially supported. While transmissibility increased with titre for isolates from two haplotypes, isolates of the H. defensa haplotype 1 were transmitted with greater frequency than isolates of other haplotypes with comparable titres. Thus, it is not sufficient to be merely frequent—endosymbionts might have to evolve specific adaptations to transmit effectively between hosts.

Endosimbiontes asociados a Diaphorina citri, vector de Candidatus Liberibacter asiaticus

iaphorina citri is considered the most dangerous citrus pest because it transmits Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing (HLB). Like other hemiptera insects, D. citri has developed mutualistic interactions with prokaryotic organisms known as endosymbionts. This symbiosis can be obligatory, when the interaction occurs with a primary endosymbiont, or facultative, when it is a secondary endosymbiont. Symbiosis is essential for various physiological functions, but some endosymbionts can adversely affect the psyllid's abilities. D. citri is associated with a great diversity of endosymbionts, with Candidatus Carsonella ruddii, Candidatus Profftella armatura, Candidatus Wolbachia spp. and Candidatus Liberibacter spp standing out. The aim o symbiotic relationship, prokaryotes, Candidatus Wolbachia, transmission of Candidatus Liberibacter asiaticus.

Whole-genome single nucleotide polymorphism and mating compatibility studies reveal the presence of distinct species in sub-Saharan Africa Bemisia tabaci whiteflies.

In sub‐Saharan Africa cassava growing areas, two members of the Bemisia tabaci species complex termed sub‐Saharan Africa 1 (SSA1) and SSA2 have been reported as the prevalent whiteflies associated with the spread of viruses that cause cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) pandemics. At the peak of CMD pandemic in the late 1990s, SSA2 was the prevalent whitefly, although its numbers have diminished over the last two decades with the resurgence of SSA1 whiteflies. Three SSA1 subgroups (SG1 to SG3) are the predominant whiteflies in East Africa and vary in distribution and biological properties. Mating compatibility between SSA1 subgroups and SSA2 whiteflies was reported as the possible driver for the resurgence of SSA1 whiteflies. In this study, a combination of both phylogenomic methods and reciprocal crossing experiments were applied to determine species status of SSA1 subgroups and SSA2 whitefly populations. Phylogenomic analyses conducted with 26 548 205 bp whole genome single nucleotide polymorphisms (SNPs) and the full mitogenomes clustered SSA1 subgroups together and separate from SSA2 species. Mating incompatibility between SSA1 subgroups and SSA2 further demonstrated their distinctiveness from each other. Phylogenomic analyses conducted with SNPs and mitogenomes also revealed different genetic relationships among SSA1 subgroups. The former clustered SSA1‐SG1 and SSA1‐SG2 together but separate from SSA1‐SG3, while the latter clustered SSA1‐SG2 and SSA1‐SG3 together but separate from SSA1‐SG1. Mating compatibility was observed between SSA1‐SG1 and SSA1‐SG2, while incompatibility occurred between SSA1‐SG1 and SSA1‐SG3, and SSA1‐SG2 and SSA1‐SG3. Mating results among SSA1 subgroups were coherent with phylogenomics results based on SNPs but not the full mitogenomes. Furthermore, this study revealed that the secondary endosymbiont—Wolbachia—did not mediate reproductive success in the crossing assays carried out. Overall, using genome wide SNPs together with reciprocal crossings assays, this study established accurate genetic relationships among cassava‐colonizing populations, illustrating that SSA1 and SSA2 are distinct species while at least two species occur within SSA1 species.

Whitefly Endosymbionts: Biology, Evolution, and Plant Virus Interactions.

Simple SummaryWhiteflies feed on plant sap and cause many problems on agricultural crops around the world. Whiteflies have endosymbiotic bacteria in cells inside their bodies that help them to feed on plants. Therefore, the sweetpotato whitefly feeds on a wide range of plants including many vegetable crops, and there are different types of these bacteria which do not all occur in the same whitefly population. In this paper, we focus on endosymbiotic bacteria that are associated with different biotypes of the sweetpotato whitefly with emphasis on their biological characteristics, diversity, and their interactions with whitefly-transmitted plant viruses. This information will be useful to the scientific community for the development of strategies to disrupt these bacteria and provide better whitefly control.Whiteflies (Hemiptera: Aleyrodidae) are sap-feeding global agricultural pests. These piercing-sucking insects have coevolved with intracellular endosymbiotic bacteria that help to supplement their nutrient-poor plant sap diets with essential amino acids and carotenoids. These obligate, primary endosymbionts have been incorporated into specialized organs called bacteriomes where they sometimes coexist with facultative, secondary endosymbionts. All whitefly species harbor the primary endosymbiont Candidatus Portiera aleyrodidarum and have a variable number of secondary endosymbionts. The secondary endosymbiont complement harbored by the cryptic whitefly species Bemisia tabaci is particularly complex with various assemblages of seven different genera identified to date. In this review, we discuss whitefly associated primary and secondary endosymbionts. We focus on those associated with the notorious B. tabaci species complex with emphasis on their biological characteristics and diversity. We also discuss their interactions with phytopathogenic begomoviruses (family Geminiviridae), which are transmitted exclusively by B. tabaci in a persistent-circulative manner. Unraveling the complex interactions of these endosymbionts with their insect hosts and plant viruses could lead to advancements in whitefly and whitefly transmitted virus management.

Diversity of Secondary Endosymbionts among Different Geographical Populations of the Grain Aphid, Sitobion avenae (Fabricius) (Hemiptera:Aphididae) in China

Aphids are one of the most challenging agricultural pests because they feed on many economic crops, and transmit pathogens including some plant viruses. The intimate associations between aphid and secondary bacterial symbionts are increasingly being recognised by significantly influencing host biology. However, related evidences, e.g., the relationship between the infected frequencies of symbionts and natural environments, have been gathered only from model pea aphids. Here, we screened for six secondary symbionts, Regiella insecticola, Hamiltonella defensa, Serratia symbiotica, Spiroplasma, Rickettsia and Arsenophonus , by using polymerase chain reaction among natural populations of the grain aphid, Sitobion avenae . Of 510 individuals from 21 localities covering the main Chinese wheat farming region have demonstrated that: i) Rickettsia was detected in all individuals, ii) R. insecticola and H. defensa showed infection frequencies of 54.7% and 7.2%, respectively, iii) S. symbiotica, Arsenophonus and Spiroplasma were not detected at all. There were different secondary endosymbionts in the same aphid populations, and multiple infections with the secondary endosymbionts: 6.7% with triple infections, 48.4% with double infections and 44.9% with single infection. Notably, the secondary endosymbionts exhibited characteristic geographical distribution patterns: Rickettsia exhibited high frequencies in the main Chinese wheat farming regions, but R. insecticola and H. defensa exhibited a remarkable infection cline. Geographic localities exhibited differences in various environmental factors that can affect the infection frequency of secondary symbionts. In the study, we found there was a statistically significant positive correlation between R. insecticola infection and environmental factors (i.e. mean annual temperature and mean annual precipitation).

Spatial Distribution of Whitefly Species (Hemiptera: Aleyrodidae) and Identification of Secondary Bacterial Endosymbionts in Tomato Fields in Costa Rica.

In Costa Rica, tomato (Solanum lycopersicum Linnaeus) Linnaeus (Solanales: Solanaceae) is one of the crops most severely affected by the whiteflies (Hemiptera: Aleyrodidae) Trialeurodes vaporariorum (Westwood) and the Bemisia tabaci (Gennadius) species complex. The objective of this study was to monitor the spatial distribution and diversity of these species and to detect the presence of secondary bacterial endosymbionts in individuals collected in areas of intensive tomato production. In total, 628 whitefly individuals were identified to the species level using restriction analysis (PCR-RFLP) of a fragment of the mitochondrial cytochrome C oxidase I gene (mtCOI). Trialeurodes vaporariorum was the predominant species, followed by B. tabaci Mediterranean (MED). Bemisia tabaci New World (NW) and B. tabaci Middle East-Asia Minor 1 (MEAM1) were present in lower numbers. The mtCOI fragment was sequenced for 89 individuals and a single haplotype was found for each whitefly species. Using molecular markers, the 628 individuals were analyzed for the presence of four endosymbionts. Arsenophonus Gherna et al. (Enterobacterales: Morganellaceae) was most frequently associated with T. vaporariorum, whereas Wolbachia Hertig (Rickettsiales: Anaplasmataceae) and Rickettsia da Rocha-Lima (Rickettsiales: Rickettsiaceae) were associated with B. tabaci MED. This study confirmed that B. tabaci NW has not been completely displaced by the invasive species B. tabaci MED and B. tabaci MEAM1 present in the country. An association was found between whitefly species present in tomato and certain secondary endosymbionts, elevation was the most likely environmental factor to affect their frequency.

Genetic diversity of Bemisia tabaci cryptic species in Nigeria and their relationships with endosymbionts and acquired begomoviruses

Abstract Bemisia tabaci is a species complex of at least 44 cryptic species with a worldwide distribution. It is a serious pest of many crop plants as well as a successful vector of at least 100 begomoviruses. Using B. tabaci collected from cassava and tomato fields in the southwestern and north central regions of Nigeria, we determined nucleotide sequences from the mitochondrial cytochrome c oxidase subunit I (COI) of 23 B. tabaci samples, the 16S and 23S ribosomal DNA of endosymbionts, and the coat protein gene of geminiviruses ingested by the whiteflies. The COI analysis identified three different genetic groups including the indigenous Sub-Saharan Africa 1 subgroup 1 (SSA1-SG1) and 5 (SSA1-SG5, which was most prevalent), and an invasive cryptic species (Mediterranean). SSA1 was infected by five known secondary endosymbionts, Arsenophonus, Cardinium, Hamiltonella, Rickettsia, and Wolbachia, and co-infections with two or three endosymbionts were common. Five begomoviruses, okra enation leaf curl virus, squash leaf curl China virus, tobacco curly shoot virus, tomato leaf curl New Delhi virus, and tomato yellow leaf curl virus, were detected from 43.5% of the B. tabaci samples. However, cassava mosaic disease that causes devastating cassava yield losses was not detected in this study. This study improves the current understanding of the genetic diversity of B. tabaci cryptic species, and it reveals their relationships with endosymbionts and geminiviruses in the cassava and tomato fields of Nigeria.

Nutritional Relationship between Bemisia tabaci and Its Primary Endosymbiont, Portiera aleyrodidarum, during Host Plant Acclimation.

Simple SummaryPlant sap-sucking insects commonly have established mutualistic relationships with bacteria that live within their bodies and often provide nutrients that are lacking in the insect’s diet. The sweet potato whitefly (Bemisia tabaci) harbors one primary and up to seven secondary endosymbiotic bacteria. The primary endosymbiont of B. tabaci is already known to play a critical role in providing necessary nutrients for B. tabaci. Our objective was to study the relationship among B. tabaci, its primary endosymbiont, and the host plant through the effects of host plant shifting and acclimation, that is, physiological adjustments as an insect becomes accustomed to a new host plant over several generations. The results showed that host shifting from Chinese kale to cotton plants led to a decrease in the fecundity of B. tabaci in the first generation, which was restored after 10 generations of acclimation, and that its developmental time was also decreased by the tenth generation. Furthermore, essential amino acid biosynthesis genes of its primary endosymbiont were differentially regulated after B. tabaci had become acclimated to cotton plants. We speculate that the primary endosymbiont has a close nutritional relationship with B. tabaci during host plant acclimation.Plant sap-sucking insects commonly have established mutualistic relationships with endosymbiotic bacteria that can provide nutrients lacking in their diet. Bemisia tabaci harbors one primary endosymbiont, Portiera aleyrodidarum, and up to seven secondary endosymbionts, including Hamiltonella defensa and Rickettsia sp. Portiera aleyrodidarum is already known to play a critical role in providing necessary nutrients for B. tabaci. In the present study, the relationship among B. tabaci, its primary endosymbiont, and the host plant were examined through the effects of host plant shifting and acclimation. Bemisia tabaci was transferred from Chinese kale to four different host plants, and the effects on both its performance and the expression levels of nutrient-related genes of P. aleyrodidarum were analyzed. The results showed that host shifting from Chinese kale to cotton plants led to a decrease in the performance of B. tabaci in the first generation, which was restored after 10 generations of acclimation. Furthermore, the expression levels of essential amino acid biosynthesis genes of P. aleyrodidarum were found to be differentially regulated after B. tabaci had acclimated to the cotton plants. Host plant shifting and acclimation to cucumber, poinsettia, and tomato plants did not affect the fecundity of B. tabaci and the expression levels of most examined genes. We speculate that P. aleyrodidarum may help B. tabaci improve its performance and acclimate to new hosts and that P. aleyrodidarum has a close nutritional relationship with its host during host plant acclimation.

The brown clock: circadian rhythms in stramenopiles.

Circadian clocks allow organisms to anticipate environmental changes associated with the diurnal light/dark cycle. Circadian oscillators have been described in plants and green algae, cyanobacteria, animals and fungi, however, little is known about the circadian clocks of photosynthetic eukaryotes outside the green lineage. Stramenopiles are a diverse group of secondary endosymbionts whose plastid originated from a red alga. Photosynthetic stramenopiles, which include diatoms and brown algae, play key roles in biogeochemical cycles and are important components of marine ecosystems. Genome annotation efforts indicated the presence of a novel type of oscillator in these organisms and the first circadian clock component in a stramenopile has been recently discovered. This review summarizes the phenotypic characterization of circadian rhythms in stramenopiles and current efforts to determine the mechanisms of this 'brown clock'. The elucidation of this brown clock will enable a deeper understanding of the role of self-sustained oscillations in the adaptation to life in marine environments.

Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon merolae as revealed by drying.

Primary plastids originated from a free-living cyanobacterial ancestor and possess their own genomes-probably a few DNA copies. These genomes, which are organized in centrally located plastid nuclei (CN-type pt-nuclei), are produced from preexisting plastids by binary division. Ancestral algae with a CN-type pt-nucleus diverged and evolved into two basal eukaryotic lineages: red algae with circular (CL-type) pt-nuclei and green algae with scattered small (SN-type) pt-nuclei. Although the molecular dynamics of pt-nuclei in green algae and plants are now being analyzed, the process of the conversion of the original algae with a CN-type pt-nucleus to red algae with a CL-type one has not been studied. Here, we show that the CN-type pt-nucleus in the primitive red alga Cyanidioschyzon merolae can be changed to the CL-type by application of drying to produce slight cell swelling. This result implies that CN-type pt-nuclei are produced by compact packing of CL-type ones, which suggests that a C. merolae-like alga was the original progenitor of the red algal lineage. We also observed that the CL-type pt-nucleus has a chain-linked bead-like structure. Each bead is most likely a small unit of DNA, similar to CL-type pt-nuclei in brown algae. Our results thus suggest a C. merolae-like alga as the candidate for the secondary endosymbiont of brown algae.

Rapid and precise genome editing in a marine diatom, Thalassiosira pseudonana by Cas9 nickase (D10A)

Diatoms are dominant phytoplankton accounting for up to 20% of annual global primary production. Diatoms are also one of the first model organisms amongst secondary endosymbionts, which have a highly chimeric genome structure accommodating genes from multiple endosymbiosis partners in their evolutionary history. Despite their ecological importance and evolutionary uniqueness, molecular-level studies have lagged behind other model organisms, hindered by difficulties in controlling their ploidy and homologous recombination system. Recently, genome editing tools, such as clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and transcription activator-like effector nucleases (TALEN), have been established in some diatoms. However, the improvement of CRISPR/Cas9 precision technology in diatoms is still underway, and the experimental complexity of TALEN limits their application in diatom molecular genetics. Cas9 nickase, a mutated version of Cas9 with a paired-single guide RNAs (sgRNAs), was recently developed to introduce a precise double cut of target DNA, suppressing the off-target effects of native Cas9 nuclease. In this study, we developed the Cas9 nickase (D10A) system for genome editing of the marine diatom Thalassiosira pseudonana. Using this system, we successfully introduced mutations into a gene encoding a putative θ-type carbonic anhydrase, a recently discovered class of carbonic anhydrase widespread in Stramenopiles. The results showed that this system introduced precise and relatively short biallelic indels to the diatom genome with minimal off-target effects.

Genetic variability, community structure, and horizontal transfer of endosymbionts among three Asia II-Bemisia tabaci mitotypes in Pakistan.

Endosymbionts associated with the whitefly Bemisia tabaci cryptic species are known to contribute to host fitness and environmental adaptation. The genetic diversity and population complexity were investigated for endosymbiont communities of B. tabaci occupying different micro‐environments in Pakistan. Mitotypes of B. tabaci were identified by comparative sequence analysis of the mitochondria cytochrome oxidase I (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II‐1, ‐5, and ‐7 mitotypes of the Asia II major clade. The whitefly–endosymbiont communities were characterized based on 16S ribosomal RNA operational taxonomic unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs occurred in the Asia II‐1 and II‐7 mitotypes (r 2 = .9, p < .005), while the Asia II‐5 microbiome was less complex. The microbiome OTU groups were mitotype‐specific, clustering with a basis in phylogeographical distribution and the corresponding ecological niche of their whitefly host, suggesting mitotype‐microbiome co‐adaptation. The primary endosymbiont Portiera was represented by a single, highly homologous OTU (0%–0.67% divergence). Two of six Arsenophonus OTUs were uniquely associated with Asia II‐5 and ‐7, and one occurred exclusively in Asia II‐1, two only in Asia II‐5, and one in both Asia II‐1 and ‐7. Four other secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and Wolbachia OTUs, were found at ≤29% frequencies. The most prevalent Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency), whereas the same strain of Cardinium and Wolbachia was found in both Asia II‐1 and ‐5, and a single Hemipteriphilus OTU occurred in Asia II‐1 and ‐7. This pattern is indicative of horizontal transfer, suggestive of a proximity between mitotypes sufficient for gene flow at overlapping mitotype ecological niches.

Outbreaks of Bemisia tabaci Mediterranean species in vegetable crops in São Paulo and Paraná States, Brazil.

The whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most important agricultural pests and virus vectors worldwide. Bemisia tabaci is considered a complex of cryptic species with at least 44 species. Among them, the species Middle East-Asia Minor 1 (MEAM1, formerly B biotype) and Mediterranean (MED, formerly Q biotype) are the most important, and they have attained global status. In Brazil, MEAM1 was first reported in the 1990s and is currently the predominant species in the country, meanwhile, MED was recently reported in the South and Southeast regions and was found to be mainly associated with ornamental plants. Currently, an increasing problem in the management of whitefly infestations in greenhouses associated with bell pepper was observed in São Paulo State, Brazil. The whiteflies were collected and identified based on a microsatellite locus (primer pair BEM23F and BEM23R) and the mitochondrial cytochrome oxidase I gene followed by restriction fragment length polymorphism analysis and sequencing. We observed that MED was the predominant species collected on bell pepper, but it was also found on tomato, cucumber, eggplant, and weeds grown in greenhouses. In open field, we found MED on tomatoes, bell peppers, and eggplants. In addition, MED was identified in Goiás State in association with ornamental plants. The begomovirus Tomato severe rugose virus and the crinivirus Tomato chlorosis virus was detected on bell pepper and tomato, respectively. Only MED specimens were found associated with the virus-infected plants. Moreover, we also investigated the endosymbionts present in the MED whiteflies. The collected populations of B. tabaci MED harbored a diversity of secondary endosymbionts, with Hamiltonella (H) found predominantly in 89 specimens of the 129 tested. These results represent a new concern for Brazilian agriculture, especially for the management of the newly introduced whitefly MED species, which must be implemented to limit the spreading and establishment of this pest in different crops in this country.