From the inside out: Were the cuticular Pseudonocardia bacteria of fungus-farming ants originally domesticated as gut symbionts?

The mutualistic interaction specificity between attine ants and antibiotic-producing Actinobacteria has been controversial because Pseudonocardia strains cannot always be isolated from worker cuticles across attine ant species, while other actinobacteria can apparently replace Pseudonocardia and also inhibit growth of Escovopsis mycopathogens. Here we report that across field samples of Panamanian species: (i) Cuticular Pseudonocardia were largely restricted to species in the crown of the attine phylogeny and their appearance likely coincided with the first attines colonizing Central/North America. (ii) The phylogenetically basal attines almost always had cuticular associations with other Actinobacteria than Pseudonocardia. (iii) The sub-cuticular glands nourishing cuticular bacteria appear to be homologous throughout the phylogeny, consistent with an ancient general attine-Actinobacteria association. (iv) The basal attine species investigated always had Pseudonocardia as gut symbionts while Pseudonocardia presence appeared mutually exclusive between cuticular and gut microbiomes. (v) Gut-associated Pseudonocardia were phylogenetically ancestral while cuticular symbionts formed a derived crown group within the Pseudonocardia phylogeny. We further show that laboratory colonies often secondarily acquire cuticular Actinobacteria that they do not associate with in the field, suggesting that many previous studies were uninformative for questions of co-adaptation in the wild. An exhaustive literature survey showed that published studies concur with our present results, provided that they analyzed field colonies and that Actinobacteria were specifically isolated from worker cuticles shortly after field collection. Our results offer several testable hypotheses for a better overall understanding of attine-Pseudonocardia interaction dynamics and putative coevolution throughout the Americas.

The role of secondary structures in the functioning of 3' untranslated regions of mRNA: A review of functions of 3' UTRs' secondary structures and hypothetical involvement of secondary structures in cytoplasmic polyadenylation in Drosophila.

3' untranslated regions (3' UTRs) of mRNAs have many functions, including mRNA processing and transport, translational regulation, and mRNA degradation and stability. These different functions require cis-elements in 3' UTRs that can be either sequence motifs or RNA structures. Here we review the role of secondary structures in the functioning of 3' UTRs and discuss some of the trans-acting factors that interact with these secondary structures in eukaryotic organisms. We propose potential participation of 3'-UTR secondary structures in cytoplasmic polyadenylation in the model organism Drosophila melanogaster. Because the secondary structures of 3' UTRs are essential for post-transcriptional regulation of gene expression, their disruption leads to a wide range of disorders, including cancer and cardiovascular diseases. Trans-acting factors, such as STAU1 and nucleolin, which interact with 3'-UTR secondary structures of target transcripts, influence the pathogenesis of neurodegenerative diseases and tumor metastasis, suggesting that they are possible therapeutic targets.

3'UTR of mRNA Encoding CPEB Protein Orb2 Plays an Essential Role in Intracellular Transport in Neurons.

Intracellular trafficking plays a critical role in the functioning of highly polarized cells, such as neurons. Transport of mRNAs, proteins, and other molecules to synaptic terminals maintains contact between neurons and ensures the transmission of nerve impulses. Cytoplasmic polyadenylation element binding (CPEB) proteins play an essential role in long-term memory (LTM) formation by regulating local translation in synapses. Here, we show that the 3'UTR of the Drosophila CPEB gene orb2 is required for targeting the orb2 mRNA and protein to synapses and that this localization is important for LTM formation. When the orb2 3'UTR is deleted, the orb2 mRNAs and proteins fail to localize in synaptic fractions, and pronounced LTM deficits arise. We found that the phenotypic effects of the orb2 3'UTR deletion were rescued by introducing the 3'UTR from the orb, another Drosophila CPEB gene. In contrast, the phenotypic effects of the 3'UTR deletion were not rescued by the 3'UTR from one of the Drosophila α-tubulin genes. Our results show that the orb2 mRNAs must be targeted to the correct locations in neurons and that proper targeting depends upon sequences in the 3'UTR.

Long-Term Memory Formation in Drosophila Depends on the 3'UTR of CPEB Gene orb2.

Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. We found that a deletion of the 3'UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, we detected more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain. Importantly, deletion of the 3'UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3'UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.

Phylogenomic analysis and metabolic role reconstruction of mutualistic Rhizobiales hindgut symbionts of Acromyrmex leaf-cutting ants.

Rhizobiales are well-known plant-root nitrogen-fixing symbionts, but the functions of insect-associated Rhizobiales are poorly understood. We obtained genomes of three strains associated with Acromyrmex leaf-cutting ants and show that, in spite of being extracellular gut symbionts, they lost all pathways for essential amino acid biosynthesis, making them fully dependent on their hosts. Comparison with 54 Rhizobiales genomes showed that all insect-associated Rhizobiales lost the ability to fix nitrogen and that the Acromyrmex symbionts had exceptionally also lost the urease genes. However, the Acromyrmex strains share biosynthesis pathways for riboflavin vitamin, queuosine and a wide range of antioxidant enzymes likely to be beneficial for the ant fungus-farming symbiosis. We infer that the Rhizobiales symbionts catabolize excess of fungus-garden-derived arginine to urea, supplementing complementary Mollicutes symbionts that turn arginine into ammonia and infer that these combined symbiont activities stabilize the fungus-farming mutualism. Similar to the Mollicutes symbionts, the Rhizobiales species have fully functional CRISPR/Cas and R-M phage defenses, suggesting that these symbionts are important enough for the ant hosts to have precluded the evolution of metabolically cheaper defenseless strains.

The 3'UTR of the Drosophila CPEB translation factor gene orb2 plays a crucial role in spermatogenesis.

CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process, orb2 mRNA and protein are localized within the developing spermatid. To evaluate the role of the orb2 mRNA 3'UTR in spermatogenesis, we used the CRISPR/Cas9 system to generate a deletion of the orb2 3'UTR, orb2R. This deletion disrupts the process of spermatid differentiation but has no apparent effect on meiosis. Differentiation abnormalities include defects in the initial polarization of the 64-cell spermatid cysts, mislocalization of mRNAs and proteins in the elongating spermatid tails, altered morphology of the elongating spermatid tails, and defects in the assembly of the individualization complex. These disruptions in differentiation appear to arise because orb2 mRNA and protein are not properly localized within the 64-cell spermatid cyst.

The role of CPEB family proteins in the nervous system function in the norm and pathology.

Posttranscriptional gene regulation includes mRNA transport, localization, translation, and regulation of mRNA stability. CPEB (cytoplasmic polyadenylation element binding) family proteins bind to specific sites within the 3'-untranslated region and mediate poly- and deadenylation of transcripts, activating or repressing protein synthesis. As part of ribonucleoprotein complexes, the CPEB proteins participate in mRNA transport and localization to different sub-cellular compartments. The CPEB proteins are evolutionarily conserved and have similar functions in vertebrates and invertebrates. In the nervous system, the CPEB proteins are involved in cell division, neural development, learning, and memory. Here we consider the functional features of these proteins in the nervous system of phylogenetically distant organisms: Drosophila, a well-studied model, and mammals. Disruption of the CPEB proteins functioning is associated with various pathologies, such as autism spectrum disorder and brain cancer. At the same time, CPEB gene regulation can provide for a recovery of the brain function in patients with fragile X syndrome and Huntington's disease, making the CPEB genes promising targets for gene therapy.

Gut microbial compositions mirror caste-specific diets in a major lineage of social insects.

Social insects owe their ecological success to the division of labour between castes, but associations between microbial community compositions and castes with different tasks and diets have not been extensively explored. Fungus-growing termites associate with fungi to degrade plant material, complemented by diverse gut microbial communities. Here, we explore whether division of labour and accompanying dietary differences between fungus-growing termite castes are linked to gut bacterial community structure. Using amplicon sequencing, we characterize community compositions in sterile (worker and soldier) and reproductive (queen and king) termites and combine this with gut enzyme activities and microscopy to hypothesise sterile caste-specific microbiota roles. Gut bacterial communities are structured primarily according to termite caste and genus and, in contrast to the observed rich and diverse sterile caste microbiotas, royal pair guts are dominated by few bacterial taxa, potentially reflecting their specialized uniform diet and unique lifestyle.

Reconstructing the functions of endosymbiotic Mollicutes in fungus-growing ants.

Mollicutes, a widespread class of bacteria associated with animals and plants, were recently identified as abundant abdominal endosymbionts in healthy workers of attine fungus-farming leaf-cutting ants. We obtained draft genomes of the two most common strains harbored by Panamanian fungus-growing ants. Reconstructions of their functional significance showed that they are independently acquired symbionts, most likely to decompose excess arginine consistent with the farmed fungal cultivars providing this nitrogen-rich amino-acid in variable quantities. Across the attine lineages, the relative abundances of the two Mollicutes strains are associated with the substrate types that foraging workers offer to fungus gardens. One of the symbionts is specific to the leaf-cutting ants and has special genomic machinery to catabolize citrate/glucose into acetate, which appears to deliver direct metabolic energy to the ant workers. Unlike other Mollicutes associated with insect hosts, both attine ant strains have complete phage-defense systems, underlining that they are actively maintained as mutualistic symbionts.

Diversity and Transmission of Gut Bacteria in Atta and Acromyrmex Leaf-Cutting Ants during Development.

The social Hymenoptera have distinct larval and adult stages separated by metamorphosis, which implies striking remodeling of external and internal body structures during the pupal stage. This imposes challenges to gut symbionts as existing cultures are lost and may or may not need to be replaced. To elucidate the extent to which metamorphosis interrupts associations between bacteria and hosts, we analyzed changes in gut microbiota during development and traced the transmission routes of dominant symbionts from the egg to adult stage in the leaf-cutting ants Acromyrmex echinatior and Atta cephalotes, which are both important functional herbivores in the New World tropics. Bacterial density remained similar across the developmental stages of Acromyrmex, but Atta brood had very low bacterial prevalences suggesting that bacterial gut symbionts are not actively maintained. We found that Wolbachia was the absolute dominant bacterial species across developmental stages in Acromyrmex and we confirmed that Atta lacks Wolbachia also in the immature stages, and had mostly Mollicutes bacteria in the adult worker guts. Wolbachia in Acromyrmex appeared to be transovarially transmitted similar to transmission in solitary insects. In contrast, Mollicutes were socially transmitted from old workers to newly emerged callows. We found that larval and pupal guts of both ant species contained Pseudomonas and Enterobacter bacteria that are also found in fungus gardens, but hardly or not in adult workers, suggesting they are beneficial only for larval growth and development. Our results reveal that transmission pathways for bacterial symbionts may be very different both between developmental stages and between sister genera and that identifying the mechanisms of bacterial acquisition and loss will be important to clarify their putative mutualistic functions.

Assessment of Chicken-Egg Membrane as a Dressing for Wound Healing.

OBJECTIVES: To examine the efficacy of the folk remedy of chicken-egg membrane dressing on wound healing. DESIGN: Full-thickness excisional wounds were created on 14 male Sprague-Dawley rats in 2 separate trials. Each animal received 2 wounds on the upper back. One wound was untreated, and the other was dressed with chicken-egg membrane to assess its impact on wound healing. Half of the rats received egg membrane treatment on the inferior wound, whereas the other half received egg membrane treatment on the superior wound. Membrane replacement, wound debridement, and imaging were done on days 5, 8, and 10 and then imaging continued on days 12, 14, 16, 18, and 20 of the experiment. Healing rate was measured based on the wound area over the 20 days of the experiment. RESULTS: The wounds dressed with chicken-egg membrane had a significantly (P < .01) faster rate of healing compared with the control at the early stages of healing between days 0 and 5. This group healed 21% faster during this early phase, compared with the control group. Overall, however, wound healing rates were indistinguishable from days 5 to 20. CONCLUSION: Chicken-egg membrane dressing significantly improves healing of cutaneous wounds in the early stages of wound healing.

Hemozoin is a product of heme detoxification in the gut of the most medically important species of the family Opisthorchiidae.

Many species of trematodes such as Schistosoma spp., Fasciola hepatica and Echinostoma trivolvis are blood-feeding parasites. Nevertheless, there is no consensus on the feeding habits of the family Opisthorchiidae (Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis). Previously, histological studies of O. felineus and C. sinensis revealed some dark stained material in their gut lumen. In this study we conducted a comprehensive analysis of the gut contents of three members of the family Opisthorchiidae (O. felineus, O. viverrini and C. sinensis). Using transmission electron microscopy, we demonstrated for the first known time the presence of disintegrating blood cells in the gut of O. felineus as well as electron-dense crystals in the gut of O. felineus and C. sinensis. Electron energy loss spectroscopy revealed iron atoms in these crystals, and mass spectrometry of the purified pigment demonstrated the presence of heme. Fourier-transform infrared spectroscopy identified the signature peaks of the common iron-carboxylate bond characteristic in crystals isolated from O. felineus and C. sinensis. Scanning electron microscopy showed layered ovoid crystals of various sizes from 50 nm to 2 µm. Morphological, chemical and paramagnetic properties of these crystals were similar to those of hemozoin from Schistosoma mansoni. Crystal formation occurs on the surface of lipid droplets in O. felineus and C. sinensis guts. Our results suggest that the diet of O. felineus and C. sinensis includes blood. Detoxification of the free heme produced during the digestion proceeds via formation of insoluble crystals that contain iron and heme dimers, i.e. crystals of hemozoin. Furthermore, we believe that biocrystallisation of hemozoin takes place on the surface of the lipid droplets, similar to S. mansoni. Hemozoin was not detected in the closely related species O. viverrini.

Acromyrmex Leaf-Cutting Ants Have Simple Gut Microbiota with Nitrogen-Fixing Potential.

Ants and termites have independently evolved obligate fungus-farming mutualisms, but their gardening procedures are fundamentally different, as the termites predigest their plant substrate whereas the ants deposit it directly on the fungus garden. Fungus-growing termites retained diverse gut microbiota, but bacterial gut communities in fungus-growing leaf-cutting ants have not been investigated, so it is unknown whether and how they are specialized on an exclusively fungal diet. Here we characterized the gut bacterial community of Panamanian Acromyrmex species, which are dominated by only four bacterial taxa: Wolbachia, Rhizobiales, and two Entomoplasmatales taxa. We show that the Entomoplasmatales can be both intracellular and extracellular across different gut tissues, Wolbachia is mainly but not exclusively intracellular, and the Rhizobiales species is strictly extracellular and confined to the gut lumen, where it forms biofilms along the hindgut cuticle supported by an adhesive matrix of polysaccharides. Tetracycline diets eliminated the Entomoplasmatales symbionts but hardly affected Wolbachia and only moderately reduced the Rhizobiales, suggesting that the latter are protected by the biofilm matrix. We show that the Rhizobiales symbiont produces bacterial NifH proteins that have been associated with the fixation of nitrogen, suggesting that these compartmentalized hindgut symbionts alleviate nutritional constraints emanating from an exclusive fungus garden diet reared on a substrate of leaves.

Ultrastructure of spermatozoa in the seminal receptacle of the liver fluke Opisthorchis felineus (Rivolta, 1884).

The spermatozoon ultrastructure in the seminal receptacle of the liver fluke Opisthorchis felineus (Digenea, Opisthorchiidae), the agent of human opisthorchiasis endemic to Russia and Eastern Europe, was examined. The bean-shaped seminal receptacle of O. felineus has a wall consisting of epithelial and muscle layers. Mature spermatozoa are located in the interior of the seminal receptacle, whereas vacuoles containing degenerating spermatozoa are detectable at the periphery. The mature spermatozoon of O. felineus has two axonemes of a 9 + "1" pattern, a nucleus, two mitochondria, a lamellar body, two bundles of parallel cortical microtubules and an external ornamentation of the plasma membrane in the anterior area of the sperm. The lamellar body is likely to be the third mitochondrion of a small size. The cytoplasm of the spermatozoon is filled with numerous electron-dense granules of storage polysaccharides. Additionally, the ultrastructural characteristics of the seminal receptacle and spermatozoa of O. felineus were compared to available published data on other trematode species. The functional roles of the observed structures of this spermatozoon are discussed.

The virulent Wolbachia strain wMelPop increases the frequency of apoptosis in the female germline cells of Drosophila melanogaster.

BACKGROUND: Wolbachia are bacterial endosymbionts of many arthropod species in which they manipulate reproductive functions. The distribution of these bacteria in the Drosophila ovarian cells at different stages of oogenesis has been amply described. The pathways along which Wolbachia influences Drosophila oogenesis have been, so far, little studied. It is known that Wolbachia are abundant in the somatic stem cell niche of the Drosophila germarium. A checkpoint, where programmed cell death, or apoptosis, can occur, is located in region 2a/2b of the germarium, which comprises niche cells. Here we address the question whether or not the presence of Wolbachia in germarium cells can affect the frequency of cyst apoptosis in the checkpoint. RESULTS: Our current fluorescent microscopic observations showed that the wMel and wMelPop strains had different effects on female germline cells of D. melanogaster. The Wolbachia strain wMel did not affect the frequency of apoptosis in cells of the germarium. The presence of the Wolbachia strain wMelPop in the D. melanogasterw1118 ovaries increased the number of germaria where cells underwent apoptosis in the checkpoint. Based on the appearance in the electron microscope, there was no difference in morphological features of apoptotic cystocytes between Wolbachia-infected and uninfected flies. Bacteria with normal ultrastructure and large numbers of degenerating bacteria were found in the dying cyst cells. CONCLUSIONS: Our current study demonstrated that the Wolbachia strain wMelPop affects the egg chamber formation in the D. melanogaster ovaries. This led to an increase in the number of germaria containing apoptotic cells. It is suggested that Wolbachia can adversely interfere either with the cystocyte differentiation into the oocyte or with the division of somatic stem cells giving rise to follicle cells and, as a consequence, to improper ratio of germline cells to follicle cells and, ultimately, to apoptosis of cysts. There was no similar adverse effect in D. melanogaster Canton S infected with the Wolbachia strain wMel. This was taken to mean that the observed increase in frequency of apoptosis was not the general effect of Wolbachia on germline cells of D. melanogaster, it was rather induced by the virulent Wolbachia strain wMelPop.

A feedback loop between Wolbachia and the Drosophila gurken mRNP complex influences Wolbachia titer.

Although much is known about interactions between bacterial endosymbionts and their hosts, little is known concerning the host factors that influence endosymbiont titer. Wolbachia endosymbionts are globally dispersed throughout most insect species and are the causative agent in filarial nematode-mediated disease. Our investigation indicates that gurken (grk), a host gene encoding a crucial axis determinant, has a cumulative, dosage-sensitive impact on Wolbachia growth and proliferation during Drosophila oogenesis. This effect appears to be mediated by grk mRNA and its protein-binding partners Squid and Hrp48/Hrb27C, implicating the grk mRNA-protein (mRNP) complex as a rate-limiting host factor controlling Wolbachia titer. Furthermore, highly infected flies exhibit defects that match those occurring with disruption of grk mRNPs, such as nurse cell chromatin disruptions and malformation of chorionic appendages. These findings suggest a feedback loop in which Wolbachia interaction with the grk mRNP affects both Wolbachia titer and grk mRNP function.