Morphometric Variation in the Hypotrich Ciliate Genus Euplotes*†

SYNOPSIS. A quantitative method for the analysis of cirral patterns in the genus Euplotes is developed and contrasted with alternative approaches. Inherent variations in cirral pattern, as they are reflected in frequency distributions of intercirral distances, are ascertained through subclonal analyses of Euplotes harpa samples grown under different conditions. This assessment of the extent of variation within a form having 10 frontoventral cirri provides a basis for a comparative study of other Euplotes of cirrotype 10. These results suggest that all marine cirrotype 10 forms with a single dargyrome have the same configuration of frontoventral and transverse cirri. Similarly, most marine double dargyrome forms of cirrotype 10 have this same cirral pattern, and thus constitute variations upon the same morphometric theme.     

Euplotes raikovi Agamaliev, 1966 (Ciliophora, Hypotrichida) From New Hampshire: Description and Morphogenesis

Euplotes raikovi, an interstitial hypotrich ciliate described once from the Caspian Sea, was isolated from intertidal sand at Rye Harbor, New Hampshire. Specimens were observed in life, and also stained by Corliss’ modification of the Chatton-Lwoff wet-silver technic and by 2 nigrosin methods. Living individuals and those fixed with Parducz's fluid are 43 × 30 (37-50 × 25–35) μm. The AZM has an average length of 27 μm and contains 24–32 membranelles. The anterior part of AZM lies on the ventral face of an apical channel, much as in E. bisulcatus. There are 7 fronto-ventral, 4 transverse, 1 left marginal, and 2 right caudal cirri. An additional small, rounded argentophilic area resembling a cirrus base is evident in silver-stained preparations, but it is barren in virtually 100% of the population. There are 7–8 (usually 7) dorsal ciliary rows with E. patella-type argyrome. The modal number of cilia in rows I-VII are 3-7-9-9-9-10-10. The unique fronto-ventral cirrus pattern is stable and predictable at the time of streak phase. Morphogenetic development indicates that the barren cirrus base is 2/V (Wallengren system), and that it apparently buds from 1/V. The left marginal cirrus and right caudal cirri have different origins.     

Description of two new species of Euplotes and Euplotes rariseta from Antarctica

Specimens of Euplotes were collected from Ross Sea (Antarctica), allowed to multiply in the laboratory, and taxonomically studied on the basis of classical diagnostic traits as seen under the optical and scanning electron microscope. They were assigned to three different morphospecies, all characterized by a dorsal silver-line system of the double type and a set of 10 fronto-ventral cirri. Two morphospecies were identified as new and named E. nobilii and E. euryhalinus; one was judged reconcilable with E. rariseta.     

Protection against a fungal pathogen conferred by the aphid facultative endosymbionts Rickettsia and Spiroplasma is expressed in multiple host genotypes and species and is not influenced by co‐infection with another symbiont

Many insects harbour facultative endosymbiotic bacteria, often more than one type at a time. These symbionts can have major effects on their hosts' biology, which may be modulated by the presence of other symbiont species and by the host's genetic background. We investigated these effects by transferring two sets of facultative endosymbionts (one Hamiltonella and Rickettsia, the other Hamiltonella and Spiroplasma) from naturally double‐infected pea aphid hosts into five novel host genotypes of two aphid species. The symbionts were transferred either together or separately. We then measured aphid fecundity and susceptibility to an entomopathogenic fungus. The pathogen‐protective phenotype conferred by the symbionts Rickettsia and Spiroplasma varied among host genotypes, but was not influenced by co‐infection with Hamiltonella. Fecundity varied across single and double infections and between symbiont types, aphid genotypes and species. Some host genotypes benefit from harbouring more than one symbiont type.     

Omikron,ein essentieller Endosymbiont von Euplotes aediculatus*†

ZUSAMMENFASSUNG. Der Süßwasserciliat Euplotes aediculatus beherbergt in seinem Cytoplasma 900–1000 stäbchenförmige Symbionten, die für seine Vermehrung essentiell zu sein scheinen. Durch Wachstum in Gegenwart von Penicillin kann Euplotes von seinen Symbionten befreit werden, es führt dies jedoch stets auch zu einem Verlust der Teilungsfähigkeit. Eine Reinfektion ist bei etwa 4% der Zellen möglich, die dann nach 4–5 Tagen sich wieder zu vermehren beginnen. Die Endosymbionten wurden sowohl lichtmikroskopisch als auch elektronenmikroskopisch untersucht. In ihrer Feinstruktur und Färbbarkeit gleichen sie, wie kappa und andere von Paramecium her bekannte “killer”-Partikel, gramnegativen Bakterien. Sie unterscheiden sich von diesen Symbionten jedoch dadurch, daß ihre DNA im Cytoplasma nicht homogen verteilt ist, sondern sich in 3–9 schon lichtmikroskopisch erkennbaren Zentren konzentriert. Die in Euplotes vorkommenden Symbionten lassen keine “killer”-Aktivität erkennen. Sie werden omikron-Partikel genannt. SYNOPSIS. The fresh water ciliate Euplotes aediculatus contains in its cytoplasm 900–1000 rod-shaped symbiotes which appear to be essential for division. Growth of Euplotes in the presence of penicillin results in loss of these symbiotes and simultaneously in a loss of the ciliate's ability to divide. Reinfection with the symbiotes can be achieved in 4% of the cells which then resume growth after a lag period of 4–5 days. The endosymbiotes have been studied by light and electron microscopy. In their fine structure and staining reaction they resemble gram-negative bacteria as do kappa and other killer particles of Paramecium. The symbiotes of Euplotes, however, are unusual in that their DNA is not distributed throughout the cytoplasm but is localized in 3–9 areas (nucleoids), which are visible even in the light microscope. No killing activity seems to be associated with the symbiotes. Following the practice of referring to those endosymbiotes by Greek letters they are here designated omikron particles.     

La Réorganisation Infraciliaire au Cours de la Conjugaison et l'Origine du Primordium Buccal dans le Genre Euplotes

L'infraciliature ventrale est presque entièrement détruite lors de la conjugaison: la totalité des structures buccales, tous les cirres fronto-ventraux et transversaux, certains cirres caudaux. Le retour à l'état végétatif s'effectue en 2 étapes. Première Etape.–Différenciation d'un nouvel ensemble de cirres, dépourvu du cirre 1/I (selon la nomenclature de Wallengren); néoformation, à partir d'une ébauche située sur le territoire présomptif du cirre manquant, d'une partie des structures buccales (la moitié antérieure de la frange adorale de membranelles). Deuxième Etape.–Remplacement de tous les cirres par un nouvel ensemble comportant le cirre 1/I; différenciation des structures buccales manquantes (moitié postérieure de la frange adorale, ciliature parorale) Les rapports morphogénétiques entre le cirre 1/I et la ciliature parorale suggèrent que le territoire de ce “cirre paroral” est homologue des territoires stomatogènes d'autres Hypotriches tels que Kahliella et Stylonychia. SYNOPSIS. During conjugation of Euplotes, the ventral ciliature, including the entire oral apparatus, all the fronto-ventral and transverse cirri, and some of the caudal cirri, is nearly completely lost. As followed in silver-stained preparations, the redifferentiation of the ciliature proceeds in 2 steps. The first step entails differentiation of a new complement of cirri, except for cirrus 1/I (according to the nomenclature of Wallengren), and neoformation of the anterior part of the adoral zone of membranelles (AZM) from a primordium located in an area that would be expected to give rise to the missing 1/I cirrus. The 2nd step involves replacement of all the cirri, including 1/I, and completion of the oral apparatus by redifferentiation of the posterior half of the AZM and of the paroral ciliature. The spatial morphogenetic relationships between cirrus 1/I and the paroral ciliature suggest that the area of this “paroral cirrus” is homologous with the stomatogenic areas of other Hypotrichida, such as Kahliella and Stylonychia.     

Happens in the best of subfamilies: establishment and repeated replacements of co‐obligate secondary endosymbionts within Lachninae aphids

Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. Most aphids from the Lachninae subfamily have been consistently found to house additional endosymbionts, mainly Serratia symbiotica. This apparent dependence on secondary endosymbionts was proposed to have been triggered by the loss of the riboflavin biosynthetic capability by Buchnera in the Lachninae last common ancestor. However, an integral large‐scale analysis of secondary endosymbionts in the Lachninae is still missing, hampering the interpretation of the evolutionary and genomic analyses of these endosymbionts. Here, we analysed the endosymbionts of selected representatives from seven different Lachninae genera and nineteen species, spanning four tribes, both by FISH (exploring the symbionts’ morphology and tissue tropism) and 16S rRNA gene sequencing. We demonstrate that all analysed aphids possess dual symbiotic systems, and while most harbour S. symbiotica, some have undergone symbiont replacement by other phylogenetically‐distinct bacterial taxa. We found that these secondary associates display contrasting cell shapes and tissue tropism, and some appear to be lineage‐specific. We propose a scenario for symbiont establishment in the Lachninae, followed by changes in the symbiont's tissue tropism and symbiont replacement events, thereby highlighting the extraordinary versatility of host‐symbiont interactions.     

Cryptic diversity,reproductive isolation and cytoplasmic incompatibility in a classic biological control success story

Molecular genetics and symbiont diagnostics have revolutionized our understanding of insect species diversity, and the transformative effects of bacterial symbionts on host life history. Encarsia inaron is a parasitoid wasp that has been shown to harbour two bacterial endosymbionts, Wolbachia and Cardinium. Known then as E. partenopea, it was introduced to the USA in the late 1980s from populations collected in Italy and Israel for the biological control of an ornamental tree pest, the ash whitefly, Siphoninus phillyreae. We studied natural populations from sites in the USA, the Mediterranean and the Middle East as well as from a Cardinium‐infected laboratory culture established from Italy, with the aims of characterizing these populations genetically, testing reproductive isolation, determining symbiont infection status in their native and introduced range, and determining symbiont role. The results showed that the two Encarsia populations introduced to the USA are genetically distinct, reproductively isolated, have different symbionts and different host–symbiont interactions, and can be considered different biological species. One (‘E. inaron’) is doubly infected by Wolbachia and Cardinium, while only Cardinium is present in the other (‘E. partenopea’). The Cardinium strains in the two species are distinct, although closely related, and crossing tests indicate that the Cardinium infecting ‘E. partenopea’ induces cytoplasmic incompatibility. The frequency of symbiont infection found in the native and introduced range of these wasps was similar, unlike the pattern seen in some other systems. These results also lead to a retelling of a successful biological control story, with several more characters than had been initially described.     

Real-time PCR reveals a high incidence of <Emphasis Type="Italic">Symbiodinium </Emphasis>clade D at low levels in four scleractinian corals across the Great Barrier Reef: implications for symbiont shuffling

Reef corals form associations with an array of genetically and physiologically distinct endosymbionts from the genus Symbiodinium. Some corals harbor different clades of symbionts simultaneously, and over time the relative abundances of these clades may change through a process called symbiont shuffling. It is hypothesized that this process provides a mechanism for corals to respond to environmental threats such as global warming. However, only a minority of coral species have been found to harbor more than one symbiont clade simultaneously and the current view is that the potential for symbiont shuffling is limited. Using a newly developed real-time PCR assay, this paper demonstrates that previous studies have underestimated the presence of background symbionts because of the low sensitivity of the techniques used. The assay used here targets the multi-copy rDNA ITS1 region and is able to detect Symbiodinium clades C and D with >100-fold higher sensitivity compared to conventional techniques. Technical considerations relating to intragenomic variation, estimating copy number and non-symbiotic contamination are discussed. Eighty-two colonies from four common scleractinian species (Acropora millepora, Acropora tenuis, Stylophora pistillata and Turbinaria reniformis) and 11 locations on the Great Barrier Reef were tested for background Symbiodinium clades. Although these colonies had been previously identified as harboring only a single clade based on SSCP analyses, background clades were detected in 78% of the samples, indicating that the potential for symbiont shuffling may be much larger than currently thought.     

Isolation of omikron-endosymbionts from mass cultures of Euplotes aediculatus and characterization of their DNA

Omikron-particles were isolated from Euplotes aediculatus cell homogenates. Purified preparations were used to extract the DNA of omikron. The melting profile suggests a GC content of 47.5% for omikron DNA. This value is 2.8% lower if it is calculated from buoyant density which was found to be 1.704 g/cm³. Similar experiments suggest a GC content of 33% for Euplotes DNA. Omikron has a DNA content of ˜3.5 × 10⁹ D per particle, and of ˜0.5 × 10⁹ D per nucleoid, since the DNA is organized in 3–9 distinct nucleoids. The kinetic complexity of omikron DNA as derived from renaturation kinetics is 0.64 × 10⁹ D and therefore by a factor of four smaller than the genome of Escherichia coli. The genome size is similar to that found for lambda-particles of Paramecium aurelia and for certain mycoplasmas.     

Faithful vertical transmission but ineffective horizontal transmission of bacterial endosymbionts during sexual reproduction of the black bean aphid,Aphis fabae

1. Insects are commonly infected with bacterial endosymbionts. In addition to the costs and benefits associated with harbouring these symbionts, their rates of vertical and horizontal transmission are important determinants of symbiont prevalence. 2. Aphids are cyclical parthenogens and show virtually perfect maternal transmission of endosymbionts during asexual reproduction. Less clear is the role of the annual sexual generation, during which overwintering eggs are produced. Data from pea aphids (Acyrthosiphon pisum Harris) suggest that maternal transmission failures and horizontal transmission via males may occur under sexual reproduction at least occasionally. No such data exist for other aphid species. 3. In the present study, the rates of maternal and paternal transmission of facultative endosymbionts during sexual reproduction in the black bean aphid, Aphis fabae (Scopoli) were examined. Crosses were performed between clones infected with Hamiltonella defensa, clones infected with Regiella insecticola and clones without facultative endosymbionts, and eggs were overwintered under three different conditions. 4. Only one of 205 offspring from crosses testing for maternal transmission failed to inherit the symbiont present in the maternal clone, and in crosses testing for horizontal transmission, only one of 412 offspring acquired a facultative symbiont from the father. 5. These results show that in A. fabae, maternal transmission of H. defensa and R. insecticola is extremely reliable also during sexual reproduction, indicating that maternal transmission failures are unlikely to exert a significant influence on frequencies of infection in the field. Paternal transmission of endosymbionts was exceedingly rare, suggesting that this route of horizontal transmission may be less important than hitherto assumed.     

Bacterial symbionts as mediators of ecologically important traits of insect hosts

1. Bacterial symbionts play a prominent role in insect nutritional ecology by aiding in digestion of food or providing nutrients that are limited or lacking in the diet. Thereby, endosymbionts open niches to their insect host that would otherwise be unavailable. 2. Currently, several other ecologically relevant traits mediated by endosymbionts are being investigated, including enhanced parasite resistance, enhanced heat tolerance, and influences on insect–plant interactions such as manipulation of plant physiology to the benefit of the insect. 3. Traits mediated by endosymbionts are often identified by correlative studies where traits are found to be altered in the presence of a particular symbiont. Recent developments in genomic tools offer the opportunity for studying the impact of bacteria–insect symbioses under natural conditions in a population and community ecology context. In vivo experiments specifically testing putative functions of endosymbionts in parallel to population‐level studies on the prevalence of endosymbionts allow disentangling host versus symbiont contribution to phenotypic variability observed in individuals. Effects of symbionts on host phenotype are often large and relevant to host fitness, e.g. by significantly enhancing survival or fecundity in a context‐dependent manner. 4. Predominantly vertically transmitted endosymbionts contribute to the heritable genetic variation present in a host species. Phenotypic variation on which selection can act may be due to differences either among host genomes, symbiont genomes, or genotype × genotype interactions. Therefore the holobiont, i.e. the host including all symbionts, should be regarded as the unit of selection as the association between host and symbionts may affect the fitness of the holobiont depending on the environment.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

Unrelated facultative endosymbionts protect aphids against a fungal pathogen

The importance of microbial facultative endosymbionts to insects is increasingly being recognized, but our understanding of how the fitness effects of infection are distributed across symbiont taxa is limited. In the pea aphid, some of the seven known species of facultative symbionts influence their host's resistance to natural enemies, including parasitoid wasps and a pathogenic fungus. Here we show that protection against this entomopathogen, Pandora neoaphidis, can be conferred by strains of four distantly related symbionts (in the genera Regiella, Rickettsia, Rickettsiella and Spiroplasma). They reduce mortality and also decrease fungal sporulation on dead aphids which may help protect nearby genetically identical insects. Pea aphids thus obtain protection from natural enemies through association with a wider range of microbial associates than has previously been thought. Providing resistance against natural enemies appears to be a particularly common way for facultative endosymbionts to increase in frequency within host populations.     

Diversity and geographic variation of endosymbiotic bacteria in natural populations of the pea aphid (Acyrthosiphon pisum) in China

Bacterial symbionts in aphids are known to benefit the insect host and associated with aphid’s ecological adaptation. The pea aphid (Acyrthosiphon pisum), an important legume pest worldwide, carries at least eight endosymbionts, providing a model system to study insect–bacteria interactions. However, species diversity and geographic variations of endosymbionts are unknown in Chinese populations; therefore, we characterized symbiont communities and diversity of 27 pea aphid samples from 13 geographic populations of China. Via amplicon high-throughput sequencing and diagnostic PCR, we found that bacterial communities of Chinese populations were dominated by Proteobacteria and Firmicutes. Among eight known endosymbionts, five (Buchnera, Serratia, Hamiltonella, Regiella, and Rickettsia) were detected by both methods, with a specific geographical distribution. The obligate symbiont, Buchnera, was present in all aphid samples, while the four facultative symbionts showed a significant geographic variation. Each population was randomly infected with distinct endosymbionts, ranging from three to five species. Serratia and Rickettsia showed relatively higher abundance in central regions of China, Regiella was predominant in eastern and western China, whereas Hamiltonella showed an extremely low abundance and was absent in four populations. Samples grouped by altitudes showed a significant diversity difference, whereas there was no significant difference between red and green body colors. Bacterial community structures of the Chinese pea aphid populations were mainly influenced by environmental factors, other than body colors. These data can guide the development of potential biocontrol techniques against this aphid.     

Intraclonal Dimorphism of Caudal Cirri in Euplotes vannus: Cortical Determination*

SYNOPSIS. Intraclonal variation in number of right caudal cirri (RCC) occurs within some species of the hypotrichous genus Euplotes. Euplotes vannus, a marine species, may have either 2 or 3 RCC. A single clone always contains individuals of both types. The frequency of individuals of each type within a clone was found to be 0.5. This fact suggested that during division each parental cell gives rise to one daughter having 3 RCC and one having 2. Formation of RCC during division was studied in E. vannus and in E. plumipes, a fresh-water form which always has 2 RCC. The studies were made on living animals and on fixed animals stained with protargol or by the Chatton-Lwoff method. In both species, the new RCC first appear in the right dorsal kineties and later migrate to the ventral surface. The RCC for the proter develop near the parental equator while those for the opisthe form near the posterior end of the parent cell, both sets developing in close proximity to kinetosomes of the kineties. In both species the 2 dorsal kineties furthest to the right each give rise to 2 RCC, one for the proter and one for the opisthe. In E. vannus, however, the third-from-the-right dorsal kinety also produces one right caudal cirrus for the proter. Therefore, in E. vannus it is the proter which always receives 3 caudal cirri and the opisthe which gets only 2. The role of the cortex in determining these events is discussed. Two cases of abnormal caudal cirrus formation are also described. Other aspects of morphogenesis during division, not previously reported, are also presented and discussed.     

Nonrandom associations of maternally transmitted symbionts in insects: The roles of drift versus biased cotransmission and selection

Virtually all higher organisms form holobionts with associated microbiota. To understand the biology of holobionts we need to know how species assemble and interact. Controlled experiments are suited to study interactions between particular symbionts, but they only accommodate a tiny portion of the diversity within each species. Alternatively, interactions can be inferred by testing if associations among symbionts in the field are more or less frequent than expected under random assortment. However, random assortment may not be a valid null hypothesis for maternally transmitted symbionts since drift alone can result in associations. Here, we analyse a European field survey of endosymbionts in pea aphids (Acyrthosiphon pisum), confirming that symbiont associations are pervasive. To interpret them, we develop a model simulating the effect of drift on symbiont associations. We show that drift induces apparently nonrandom assortment, even though horizontal transmissions and maternal transmission failures tend to randomise symbiont associations. We also use this model in the approximate Bayesian computation framework to revisit the association between Spiroplasma and Wolbachia in Drosophila neotestacea. New field data reported here reveal that this association has disappeared in the investigated location, yet a significant interaction between Spiroplasma and Wolbachia can still be inferred. Our study confirms that negative and positive associations are pervasive and often induced by symbiont‐symbiont interactions. Nevertheless, some associations are also likely to be driven by drift. This possibility needs to be considered when performing such analyses, and our model is helpful for this purpose.     

Co-evolution and symbiont replacement shaped the symbiosis between adelgids (Hemiptera: Adelgidae) and their bacterial symbionts

The Adelgidae (Insecta: Hemiptera), a small group of insects, are known as severe pests on various conifers of the northern hemisphere. Despite of this, little is known about their bacteriocyte‐associated endosymbionts, which are generally important for the biology and ecology of plant sap‐sucking insects. Here, we investigated the adelgid species complexes Adelges laricis/tardus, Adelges abietis/viridis and Adelges cooleyi/coweni, identified based on their coI and ef1alpha genes. Each of these insect groups harboured two phylogenetically different bacteriocyte‐associated symbionts belonging to the Betaproteobacteria and the Gammaproteobacteria, respectively, as inferred from phylogenetic analyses of 16S rRNA gene sequences and demonstrated by fluorescence in situ hybridization. The betaproteobacterial symbionts of all three adelgid complexes (‘Candidatus Vallotia tarda’, ‘Candidatus Vallotia virida’ and ‘Candidatus Vallotia cooleyia’) share a common ancestor and show a phylogeny congruent with that of their respective hosts. Similarly, there is evidence for co‐evolution between the gammaproteobacterial symbionts (‘Candidatus Profftia tarda’, ‘Candidatus Profftia virida’) and A. laricis/tardus and A. abietis/viridis. In contrast, the gammaproteobacterial symbiont of A. cooleyi/coweni (‘Candidatus Gillettellia cooleyia’) is different from that of the other two adelgids but shows a moderate relationship to the symbiont ‘Candidatus Ecksteinia adelgidicola’ of A. nordmannianae/piceae. All symbionts were present in all adelgid populations and life stages analysed, suggesting vertical transmission from mother to offspring. In sharp contrast to their sister group, the aphids, adelgids do not consistently contain a single obligate (primary) symbiont but have acquired phylogenetically different bacterial symbionts during their evolution, which included multiple infections and symbiont replacement.