Tissue Localization of the Endosymbiotic Bacterium “Candidatus Blochmannia floridanus” in Adults and Larvae of the Carpenter Ant Camponotus floridanus

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed C. floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.     

Replication of the endosymbiotic bacterium Blochmannia floridanus is correlated with the developmental and reproductive stages of its ant host

The dynamics of replication of the intracellular endosymbiotic bacterium Blochmannia floridanus was determined during the larval development of its host ant Camponotus floridanus by real-time quantitative PCR. The bacteria were found to proliferate during pupation and immediately after the eclosion of the imagines (adult ants). In older workers the number of bacteria present in the midgut bacteriocytes decreased significantly. In contrast, the bacterial population in the ovaries was dependent on the reproductive state of the animal. An age-dependent degeneration of the midgut bacteriocytes was also investigated by microscopic techniques in males and female castes of the closely related ant species C. herculeanus and C. sericeiventris, respectively, with similar results and supports the concept of age-dependent degeneration of the midgut bacteriocytes in all castes.     

Tissue localization of the endosymbiotic bacterium "Candidatus Blochmannia floridanus" in adults and larvae of the carpenter ant Camponotus floridanus

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed C. floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.     

Ultrastructural Study of an Endosymbiotic Alga and Its Host Ciliate Stentor niger

Stentor niger collected in the suburbs of Hiroshima contained in its cytoplasm several hundreds of endosymbiotic algae and innumerable brownish pigment granules. The body of the ciliate was dark due to a mixture of the green endosymbiotic algae and brown pigment granules. The algae belonged to the genus Chlorella; each was enclosed in a perialgal vacuole and dispersed uniformly in the host cytoplasm from the myoneme layer inward to the center of the ciliate. The cell wall and plasma membrane of the alga enclosed a nucleus, chloroplast, mitochondrion, Golgi complex, accumulation bodies, myelinated vesicles, and many ribosomes. The chloroplast occupied more than half of the volume of the alga and contained a conspicuous pyrenoid. Algal multiplication occurred by two successive divisions of an alga, leading to four autospores within a perialgal vacuole; the walls of the vacuole invaginated to separate the autospores each into its own vacuole. Three types of pigment granules were scattered uniformly throughout the cytoplasm of the ciliate. The ultrastructure of the membranellar region, somatic cortex, and macro- and micronucleus of the ciliate are also described.     

Distribution of Endosymbiotic Reproductive Manipulators Reflects Invasion Process and Not Reproductive System Polymorphism in the Little Fire Ant Wasmannia auropunctata

Endosymbiotic reproductive manipulators may have drastic effects on the ecological and evolutionary dynamics of their hosts. The prevalence of these endosymbionts reflects both their ability to manipulate their hosts and the history of the host populations. The little fire ant Wasmannia auropunctata displays a polymorphism in both its reproductive system (sexual versus clonal populations) and the invasive status of its populations (associated to a habitat shift). We first screened for the presence of a diverse array of reproductive parasites in sexual and clonal populations of W. auropunctata, as a means to investigate the role of endosymbionts in reproductive phenotypes. Wolbachia was the only symbiont found and we then focused on its worldwide distribution and diversity in natural populations of W. auropunctata. Using a multilocus scheme, we further characterized the Wolbachia strains present in these populations. We found that almost all the native sexual populations and only a few clonal populations are infected by Wolbachia. The presence of similar Wolbachia strains in both sexual and clonal populations indicates that they are probably not the cause of the reproductive system polymorphism. The observed pattern seems rather associated to the invasion process of W. auropunctata. In particular, the observed loss of Wolbachia in clonal populations, that recurrently emerged from sexual populations, likely resulted from natural heat treatment and/or relaxed selection during the shift in habitat associated to the invasion process.     

Patterns and rates of nucleotide substitution,insertion and deletion in the endosymbiont of ants Blochmannia floridanus

Genome reduction is a general process that has been studied in numerous symbiotic bacteria associated with insects. We investigated the last stages of genome degradation in Blochmannia floridanus, a mutualistic bacterial endosymbiont of the ant Camponotus floridanus. We determined the tempo (rates of insertion and deletion) and mode (size and number of insertion–deletion events) of the process in the last 200 000 years by analysing a total of 16 intergenic regions in several strains of this endosymbiont from different ant populations. We provide the first calculation of the reduction rate for noncoding DNA in this endosymbiont (2.2 × 10^?8 lost nucleotides/site/year) and compare it with the rate of loss in other species. Our results confirm, as it has been observed in other organisms like Buchnera aphidicola or Rickettsia spp., that deletions larger than one nucleotide can still appear in advanced stages of genome reduction and that a substitutional deletion bias exists. However, this bias is not due to a higher proportion of deletion over insertion events but to a few deletion events being larger than the rest. Moreover, we detected a substitutional AT bias that is probably responsible for the increase in the number of the small and moderate indel events in the last stages of genome reduction. Accordingly, we found intrapopulational polymorphisms for the detected microsatellites in contrast to the stability associated with these in free‐living bacteria such as Escherichia coli.     

Large-Scale Intersubspecific Recombination in the Plant-Pathogenic Bacterium Xylella fastidiosa Is Associated with the Host Shift to Mulberry

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.     

Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii

P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells.     

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-¹⁴C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.     

MC1R Is a Prognostic Marker and Its Expression Is Correlated with MSI in Colorectal Cancer

Melanocortin 1 receptor (MC1R) is thought to be a marker of poor prognosis and a potential target for the treatment of melanoma. Studies have found that MC1R promotes several tumor behaviors, including cell proliferation and differentiation, pigment formation, and genome damage repair. Some single-nucleotide polymorphisms (SNPs) of MC1R are involved in the occurrence and development of melanoma. A few studies have reported a relationship between MC1R and colorectal cancer (CRC). In this research, our objective was to examine MC1R expression and MC1R SNPs and investigate their correlation with the clinicopathological features of human CRC tissues. We evaluated MC1R mRNA expression by performing bioinformatic analyses on human CRC expression datasets. We used Western blotting and RT-qPCR to compare MC1R expression in CRC tissues with that in normal tissues, and MC1R SNPs in CRC tissues were detected by PCR-direct sequencing (DS). The expression of MC1R was significantly decreased in CRC tissues compared with normal tissue, and its expression was negatively associated with P53 expression, MLH1 expression, and PMS2 expression, and high MC1R expression was significantly associated with microsatellite instability (MSI). MC1R SNPs were also associated with the clinicopathological characteristics of CRC; for example, the rs2228479 locus genotype was correlated with Ki67 status, and the rs885479 locus genotype was correlated with age and T stage. In conclusion, MC1R plays a crucial role in the progression of CRC and may be a marker of poor prognosis in CRC.     

Reduction of Tubulin Expression in Angomonas deanei by RNAi Modifies the Ultrastructure of the Trypanosomatid Protozoan and Impairs Division of Its Endosymbiotic Bacterium

In the last two decades, RNA interference pathways have been employed as a useful tool for reverse genetics in trypanosomatids. Angomonas deanei is a nonpathogenic trypanosomatid that maintains an obligatory endosymbiosis with a bacterium related to the Alcaligenaceae family. Studies of this symbiosis can help us to understand the origin of eukaryotic organelles. The recent elucidation of both the A. deanei and the bacterium symbiont genomes revealed that the host protozoan codes for the enzymes necessary for RNAi activity in trypanosomatids. Here, we tested the functionality of the RNAi machinery by transfecting cells with dsRNA to a reporter gene (green fluorescent protein), which had been previously expressed in the parasite and to α‐tubulin, an endogenous gene. In both cases, protein expression was reduced by the presence of specific dsRNA, inducing, respectively, a decreased GFP fluorescence and the formation of enlarged cells with modified arrangement of subpellicular microtubules. Furthermore, symbiont division was impaired. These results indicate that the RNAi system is active in A. deanei and can be used to further explore gene function in symbiont‐containing trypanosomatids and to clarify important aspects of symbiosis and cell evolution.     

贝氏隐孢子虫在鸡体内发育的形态学特征及其对寄生器官的影响

取 2日龄海兰雏鸡 5 0只 ,分为 5组 ,分别接种 0、 0 8× 10 6、 1 6× 10 6、 3 2× 10 6、 6 4× 10 6个鸭源贝氏隐孢子虫 (Cryptosporidiumbaileyi)卵囊。接种后在不同的时间间隔内剖杀雏鸡 ,取法氏囊、气管和喉头。扫描电镜观察发现贝氏隐孢子虫主要寄生在鸡的喉头、法氏囊、气管。裂殖体有 2种类型 :Ⅰ型裂殖体含 8个裂殖子 ,Ⅱ型裂殖体含 4个裂殖子。子孢子或裂殖子在钻入过程中 ,虫体逐渐由香蕉形过渡到鼓槌形 ,最后形成球形的滋养体。带虫空泡分为有球形残体的带虫空泡和无球形残体的带虫空泡。观察到小配子的释放和虫体寄生于杯状细胞的现象。贝氏隐孢子虫寄生于气管引起纤毛倒伏、排列紊乱、纤毛融合、脱落 ;致使法氏囊上皮肿胀 ,法氏囊粘膜表面形成皱褶 ,微绒毛脱落、融合、排列紊乱 ,粘液性分泌物增多 ,炎性细胞渗出     

Properties and Characterization of the Host Bacterium of Bacteriophage PM2

A marine bacterium, which acts as host for bacteriophage PM2 isolated from the same environment, has been characterized as a Pseudomonas species. This Pseudomonas cannot be assigned to any of the subgeneric categories or "groups" described by Stanier, Palleroni, and Doudoroff. The psychrophilic and halophilic nature of the organism and its requirement for NaCl suggest an indigenous marine origin.