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Many insects and other animals carry microbial endosymbionts that influence their reproduction and fitness. These relationships only persist if endosymbionts are reliably transmitted from one host generation to the next. Wolbachia are maternally transmitted endosymbionts found in most insect species, but transmission rates can vary across environments. Maternal transmission of wMel Wolbachia depends on temperature in natural Drosophila melanogaster hosts and in transinfected Aedes aegypti, where wMel is used to block pathogens that cause human disease. In D. melanogaster, wMel transmission declines in the cold as Wolbachia become less abundant in host ovaries and at the posterior pole plasm (the site of germline formation) in mature oocytes. Here, we assess how temperature affects maternal transmission and underlying patterns of Wolbachia localization across 10 Wolbachia strains diverged up to 50 million years-including strains closely related to wMel-and their natural Drosophila hosts. Many Wolbachia maintain high transmission rates across temperatures, despite highly variable (and sometimes low) levels of Wolbachia in the ovaries and at the developing germline in late-stage oocytes. Identifying strains like closely related wMel-like Wolbachia with stable transmission across variable environmental conditions may improve the efficacy of Wolbachia-based biocontrol efforts as they expand into globally diverse environments., (© 2024. The Author(s).)

Many animal- and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells 1,2 . Elucidation of how these effector proteins function in host cells is critical for understanding infectious diseases in animals and plants 3-5 . The widely conserved AvrE-family effectors, including DspE in Erwinia amylovora and AvrE in Pseudomonas syringae, have a central role in the pathogenesis of diverse phytopathogenic bacteria 6 . These conserved effectors are involved in the induction of 'water soaking' and host cell death that are conducive to bacterial multiplication in infected tissues. However, the exact biochemical functions of AvrE-family effectors have been recalcitrant to mechanistic understanding for three decades. Here we show that AvrE-family effectors fold into a β-barrel structure that resembles bacterial porins. Expression of AvrE and DspE in Xenopus oocytes results in inward and outward currents, permeability to water and osmolarity-dependent oocyte swelling and bursting. Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye. Targeted screening of chemical blockers based on the predicted pore size (15-20 Å) of the DspE channel identified polyamidoamine dendrimers as inhibitors of the DspE/AvrE channels. Notably, polyamidoamines broadly inhibit AvrE and DspE virulence activities in Xenopus oocytes and during E. amylovora and P. syringae infections. Thus, we have unravelled the biochemical function of a centrally important family of bacterial effectors with broad conceptual and practical implications in the study of bacterial pathogenesis., (© 2023. The Author(s).)

Purpose: Does existing scientific literature suggest an impact of oocyte dysmorphisms on biological or clinical outcomes of assisted reproduction treatments?, Methods: Studies of interest were selected from an initial cohort of 6651 potentially relevant records retrieved. PubMed was systematically searched for peer-reviewed original papers and reviews identified by keywords and medical subject heading (MeSH) terms. The most relevant publications were critically evaluated to identify criteria for oocyte morphological evaluation and IVF outcomes. For each morphological abnormality, we generated an oocyte literature score (OLS) through the following procedure: (a) papers showing a negative, absence of, or positive correlation between a given abnormality and IVF outcome were scored 1, 0, and - 1, respectively; (b) the sum of these scores was expressed as a fraction of all analyzed papers; (c) the obtained fraction was multiplied by 10 and converted into decimal number., Result: We identified eleven different dysmorphisms, of which six were extracytoplasmic (COC, zona pellucida, perivitelline space, polar body 1, shape, giant size) and five intracytoplasmic (vacuoles, refractile bodies, SER clusters, granularity, color). Among the extracytoplasmic dysmorphisms, abnormal morphology of the COC generated an OLS of 8.33, indicating a large prevalence (5/6) of studies associated with a negative outcome. Three intracytoplasmic dysmorphisms (vacuoles, SER clusters, and granularity) produced OLS of 7.14, 7.78, and 6.25, respectively, suggestive of a majority of studies reporting a negative outcome., Conclusion: COC morphology, vacuoles, SER clusters, and granularity produced OLS suggestive of a prevalence of studies reporting a negative outcome., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes., Method(s): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um 2 ), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement., Results: Male zygotes had longer time intervals of tPB2&#95;tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um 2 , p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um 2 , p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023)., Conclusion(s): Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Endosymbiont transmission via eggs to future host generations has been recognized as the main strategy for its persistence in insect hosts; however, the mechanisms for transmission have yet to be elucidated. Here, we describe the dynamic locations of Rickettsia in the ovarioles and eggs during oogenesis and embryogenesis in a globally significant pest whitefly Bemisia tabaci. Field populations of the whitefly have a high prevalence of Rickettsia, and in all Rickettsia-infected individuals, the bacterium distributes in the body cavity of the host, especially in the midgut, fat body, hemocytes, hemolymph, and near bacteriocytes. The distribution of Rickettsia was subjected to dynamic changes in the ovary during oogenesis, and our ultrastructural observations indicated that the bacteria infect host ovarioles during early developmental stages via two routes: (i) invasion of the tropharium by endocytosis and then transmission into vitellarium via nutritive cord and (ii) entry into vitellarium by hijacking bacteriocyte translocation. Most of the Rickettsia are degraded in the oocyte cytoplasm in late-stage oogenesis. However, a few reside beneath the vitelline envelope of mature eggs, spread into the embryo, and proliferate during embryogenesis to sustain high-fidelity transmission to the next generation. Our findings provide novel insights into the maternal transmission underpinning the persistence and spread of insect symbionts.

Symbiotic associations with beneficial microorganisms endow a variety of host animals with adaptability to the environment. Stable transmission of symbionts across host generations is a key event in the maintenance of symbiotic associations through evolutionary time. However, our understanding of the mechanisms of symbiont transmission remains fragmentary. The deep-sea clam Phreagena okutanii harbors chemoautotrophic intracellular symbiotic bacteria in gill epithelial cells, and depends on these symbionts for nutrition. In this study, we focused on the association of these maternally transmitted symbionts with ovarian germ cells in juvenile female clams. First, we established a sex identification method for small P. okutanii individuals, and morphologically classified female germ cells observed in the ovary. Then, we investigated the association of the endosymbiotic bacteria with germ cells. We found that the symbionts were localized on the outer surface of the cell membrane of primary oocytes and not within the cluster of oogonia. Based on our findings, we discuss the processes and mechanisms of symbiont vertical transmission in P. okutanii .

Many insect species, such as aphids, leafhoppers, planthoppers, and whiteflies harbor obligate bacterial symbionts that can be transovarially transmitted to offspring through the oocytes of female insects. Whether obligate bacterial symbionts can carry important molecules/resources to the embryos to support egg development is still unknown. Here, we show that the vitellogenin (Vg) precursor of rice leafhopper Nephotettix cincticeps is biosynthesized by the fat body, secreted into the hemolymph and subsequently cleaved into the 35- and 178-kDa subunits, whereas only the 178-kDa subunit is taken up by the leading end of oocytes in a receptor-dependent manner or moves into the posterior pole of the terminal oocyte in association with obligate bacterial symbiont " Candidatus Nasuia deltocephalinicola" (hereafter Nasuia ) in a receptor-independent manner. Furthermore, the 178-kDa Vg subunit can directly interact with a surface channel molecule (porin) on the envelope of Nasuia , allowing Vg to enter bacterial cytoplasm. Thus, Vg can hitchhike the ancient oocyte entry path of Nasuia , the common obligate symbiont of leafhoppers. Knocking down a Nasuia growth-related protein expression or treatment with porin antibody strongly prevents the ability of Nasuia to carry Vgs into oocytes and impair insect egg development. Nasuia -carried Vgs provide at least 20% of the total Vgs in the developing eggs. We anticipate that the bacterial symbiont-mediated Vg uptake into oocytes to support efficient egg development may be a common pattern shared by many insects. IMPORTANCE Many insects harbor obligate bacterial symbionts that can be vertically transmitted to offspring by female insects through eggs. Here, we report that leafhopper vitellogenin (Vg) recognizes and binds a surface channel molecule (porin) on the envelope of obligate bacterial symbiont Nasuia , which potentially induces the opening of porin channels for Vg to access the cytoplasm of Nasuia Thus, Vg can exploit bacterial symbionts as the independent carriers into the oocytes. Such Nasuia -carried Vg contents support efficient insect egg development. Thus, our findings indicate that insects have evolved strategies to exploit the symbionts for carrying additional Vgs to guarantee optimal insect reproduction., (Copyright © 2020 Mao et al.)

Whiteflies possess bacterial symbionts Candidatus Portiera aleyrodidium that are housed in specialized cells called bacteriocytes and are faithfully transmitted via the ovary to insect offspring. In one whitefly species studied previously, Bemisia tabaci MEAM1, transmission is mediated by somatic inheritance of bacteriocytes, with a single bacteriocyte transferred to each oocyte and persisting through embryogenesis to the next generation. Here, we investigate the mode of bacteriocyte transmission in two whitefly species, B. tabaci MED, the sister species of MEAM1, and the phylogenetically distant species Trialeurodes vaporariorum. Microsatellite analysis supported by microscopical studies demonstrates that B. tabaci MED bacteriocytes are genetically different from other somatic cells and persist through embryogenesis, as for MEAM1, but T. vaporariorum bacteriocytes are genetically identical to other somatic cells of the insect, likely mediated by the degradation of maternal bacteriocytes in the embryo. These two alternative modes of transmission provide a first demonstration among insect symbioses that the cellular processes underlying vertical transmission of bacterial symbionts can diversify among related host species associated with a single lineage of symbiotic bacteria., (© 2019 The Authors. Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

In our previous study, all Arthrinium isolates from Sargassum sp. showed high bioactivities, but studies on marine Arthrinium spp. are insufficient. In this study, a phylogenetic analysis of 28 Arthrinium isolates from seaweeds and egg masses of Arctoscopus japonicus was conducted using internal transcribed spacers, nuclear large subunit rDNA, β-tubulin, and translation elongation factor region sequences, and their bioactivities were investigated. They were analyzed as 15 species, and 11 of them were found to be new species. Most of the extracts exhibited radical-scavenging activity, and some showed antifungal activities, tyrosinase inhibition, and quorum sensing inhibition. It was implied that marine algicolous Arthrinium spp. support the regulation of reactive oxygen species in symbiotic algae and protect against pathogens and bacterial biofilm formation. The antioxidant from Arthrinium sp. 10 KUC21332 was separated by bioassay-guided isolation and identified to be gentisyl alcohol, and the antioxidant of Arthrinium saccharicola KUC21221 was identical. These results demonstrate that many unexploited Arthrinium species still exist in marine environments and that they are a great source of bioactive compounds., Competing Interests: The authors declare no conflict of interest.

Widespread success of the intracellular bacterium Wolbachia across insects and nematodes is due to efficient vertical transmission and reproductive manipulations. Many strains, including wMel from Drosophila melanogaster, exhibit a specific concentration to the germplasm at the posterior pole of the mature oocyte, thereby ensuring high fidelity of parent-offspring transmission. Transport of Wolbachia to the pole relies on microtubules and the plus-end directed motor kinesin heavy chain (KHC). However, the mechanisms mediating Wolbachia's association with KHC remain unknown. Here we show that reduced levels of the host canonical linker protein KLC results in dramatically increased levels of Wolbachia at the oocyte's posterior, suggesting that KLC and some key associated host cargos outcompete Wolbachia for association with a limited amount of KHC motor proteins. Consistent with this interpretation, over-expression of KHC causes similarly increased levels of posteriorly localized Wolbachia. However, excess KHC has no effect on levels of Vasa, a germplasm component that also requires KHC for posterior localization. Thus, Wolbachia transport is uniquely KHC-limited because these bacteria are likely outcompeted for binding to KHC by some host cargo/linker complexes. These results reveal a novel host-symbiont interaction that underscores the precise regulation required for an intracellular bacterium to co-opt, but not disrupt, vital host processes., Competing Interests: The authors have declared that no competing interests exist.

Anoplolepis gracilipes is one of the six most widespread and pestiferous invasive ant species. Populations of this invader in Arnhem Land, Australia have been observed to decline, but the reasons behind these declines are not known. We investigated if there is evidence of a pathogen that could be responsible for killing ant queens or affecting their reproductive output. We measured queen number per nest, fecundity and fat content of queens from A. gracilipes populations in various stages of decline or expansion. We found no significant difference in any of these variables among populations. However, 23% of queens were found to have melanized nodules, a cellular immune response, in their ovaries and fat bodies. The melanized nodules found in dissected queens are highly likely to indicate the presence of pathogens or parasites capable of infecting A. gracilipes. Queens with nodules had significantly fewer oocytes in their ovaries, but nodule presence was not associated with low ant population abundances. Although the microorganism responsible for the nodules is as yet unidentified, this is the first evidence of the presence of a pathogenic microorganism in the invasive ant A. gracilipes that may be affecting reproduction.

Within aquatic habitats, the hyper-abundant Order Crustacea appear to be the predominant host group for members of the Phylum Microsporidia. The musculature, a common site of infection, provides access to biochemical (carbohydrate-rich) and physiological (mitochondria-rich) conditions conducive to prolific parasite replication and maturation. The significant proportion of body plan devoted to skeletal musculature in Crustacea provides the location for a highly efficient intracellular parasite factory. In this study, we utilize histological, ultrastructural and phylogenetic evidence to describe a previously known (Inodosporus octospora) and novel (Ovipleistophora arlo n. sp.) microsporidian parasites infecting the musculature of the common prawn (Palaemon serratus) from the same site, at the same time of year. Despite similar clinical signs of infection, both parasites are otherwise distinct in terms of pathogenesis, morphology and phylogeny. Based upon partial subunit ribosomal RNA (SSU rDNA) sequence, we show that that I. octospora may be identical to a Kabatana sp. previously described infecting two-spot goby (Gobiusculus flavescens) in Europe, or at least that Inodosporus and Kabatana genera are synonyms. In addition, SSU rDNA sequence for O. arlo places it within a distinct clade containing Ovipleistophora mirandellae and Ovipleistophora ovariae, both infecting the oocytes of freshwater fish in Europe. Taken together, our data provide strong evidence for trophic-transfer between crustacean and fish hosts for two different microsporidians within clade 5 of the phylum. Furthermore, it demonstrates that morphologically and phylogenetically distinct microsporidians can infect the same tissues of the same host species to impart clinical signs which mimic infection with the other.

Background: Apoptosis is programmed cell death that ordinarily occurs in ovarian follicular cells in various organisms. In the best-studied holometabolous insect, Drosophila, this kind of cell death occurs in all three cell types found in the follicles, sometimes leading to follicular atresia and egg degeneration. On the other hand, egg development, quantity and viability in the mosquito Culex quinquefasciatus are disturbed by the infection with the endosymbiont Wolbachia. Considering that Wolbachia alters reproductive traits, we hypothesised that such infection would also alter the apoptosis in the ovarian cells of this mosquito. The goal of this study was to comparatively describe the occurrence of apoptosis in Wolbachia-infected and uninfected ovaries of Cx. quinquefasciatus during oogenesis and vitellogenesis. For this, we recorded under confocal microscopy the occurrence of apoptosis in all three cell types of the ovarian follicle. In the first five days of adult life we observed oogenesis and, after a blood meal, the initiation step of vitellogenesis., Results: Apoptoses in follicular cells were found at all observation times during both oogenesis and vitellogenesis, and less commonly in nurse cells and the oocyte, as well as in atretic follicles. Our results suggested that apoptosis in follicular cells occurred in greater numbers in infected mosquitoes than in uninfected ones during the second and third days of adult life and at the initiation step of vitellogenesis., Conclusions: The presence of Wolbachia leads to an increase of apoptosis occurrence in the ovaries of Cx. quinquefasciatus. Future studies should investigate if this augmented apoptosis frequency is the cause of the reduction in the number of eggs laid by Wolbachia-infected females. Follicular atresia is first reported in the previtellogenic period of oogenesis. Our findings may have implications for the use of Wolbachia as a mosquito and pathogens control strategy.

Cyst nematodes are globally important pathogens in agriculture. Their sedentary lifestyle and long-term association with the roots of host plants render cyst nematodes especially good targets for attack by parasitic fungi. In this context fungi were specifically isolated from nematode eggs of the cereal cyst nematode Heterodera filipjevi. Here, Ijuhya vitellina (Ascomycota, Hypocreales, Bionectriaceae), encountered in wheat fields in Turkey, is newly described on the basis of phylogenetic analyses, morphological characters and life-style related inferences. The species destructively parasitises eggs inside cysts of H. filipjevi. The parasitism was reproduced in in vitro studies. Infected eggs were found to harbour microsclerotia produced by I. vitellina that resemble long-term survival structures also known from other ascomycetes. Microsclerotia were also formed by this species in pure cultures obtained from both, solitarily isolated infected eggs obtained from fields and artificially infected eggs. Hyphae penetrating the eggshell colonised the interior of eggs and became transformed into multicellular, chlamydospore-like structures that developed into microsclerotia. When isolated on artificial media, microsclerotia germinated to produce multiple emerging hyphae. The specific nature of morphological structures produced by I. vitellina inside nematode eggs is interpreted as a unique mode of interaction allowing long-term survival of the fungus inside nematode cysts that are known to survive periods of drought or other harsh environmental conditions. Generic classification of the new species is based on molecular phylogenetic inferences using five different gene regions. I. vitellina is the only species of the genus known to parasitise nematodes and produce microsclerotia. Metabolomic analyses revealed that within the Ijuhya species studied here, only I. vitellina produces chaetoglobosin A and its derivate 19-O-acetylchaetoglobosin A. Nematicidal and nematode-inhibiting activities of these compounds have been demonstrated suggesting that the production of these compounds may represent an adaptation to nematode parasitism.

Our understanding of the functions of vitellogenin (Vtg) in reproduction has undergone an evolutionary transformation over the past decade. Primarily, Vtg was regarded as a female-specific reproductive protein, which is cleaved into yolk proteins including phosvitin (Pv) and lipovitellin (Lv), stored in eggs, providing the nutrients for early embryos. Recently, Vtg has been shown to be an immunocomponent factor capable of protecting the host against the attack by microbes including bacteria and viruses. Moreover, Pv and Lv that both are proteolytically cleaved products of maternal Vtg, as well as Pv-derived small peptides, all display an antibacterial role in developing embryos. In addition, both Vtg and yolk protein Pv possess antioxidant activity capable of protecting cells from damage by free radicals. Collectively, these data indicate that Vtg, in addition to being involved in yolk protein formation, also plays non-nutritional roles via functioning as immune-relevant molecules and antioxidant reagents.

The bacterial endosymbiont Blochmannia floridanus of the carpenter ant Camponotus floridanus contributes to its hosts' ontogeny via nutritional upgrading during metamorphosis. This primary endosymbiosis is essential for both partners and vertical transmission of the endosymbionts is guaranteed by bacterial infestation of oocytes. Here we present a detailed analysis of the presence and localisation of B. floridanus in the ants' ovaries obtained by FISH and TEM analyses. The most apical part of the germarium harbouring germ-line stem cells (GSCs) is not infected by the bacteria. The bacteria are detectable for the first time in lower parts of the germarium when cystocytes undergo the 4th and 5th division and B. floridanus infects somatic cells lying under the basal lamina surrounding the ovarioles. With the beginning of cystocyte differentiation, the endosymbionts are exclusively transported from follicle cells into the growing oocytes. This infestation of the oocytes by bacteria very likely involves exocytosis-endocytosis processes between follicle cells and the oocytes. Nurse cells were never found to harbour the endosymbionts. Furthermore we present first gene expression data in C. floridanus ovaries. These data indicate a modulation of immune gene expression which may facilitate tolerance towards the endosymbionts and thus may contribute to their transovarial transmission., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To study the contamination risk in open and closed vitrification devices for oocyte/embryo cryopreservation by evaluating the contaminants present (bacteria and fungi) in the thaw medium and in liquid nitrogen (LN) storage containers., Design: Retrospective study., Setting: Human reproduction unit., Patient(s): None., Intervention(s): Retrospective study of vitrification device safety and LN sterility performed from July to October 2014., Main Outcome Measure(s): From each bank container, both open and closed vitrification devices, devitrification media and LN in the containers and as supplied by the company were evaluated for contaminants. An automated system and the corresponding susceptibility to antibiotics were used for bacteria identification. Fungus detection was performed by evaluating the colony morphology and their microscopic characteristics., Result(s): No bacteria or fungi were observed in any of the devitrification media regardless of the type of device used, nor in the LN supplied by the company. No fungi were observed in any of the LN samples tested. Stenotrophomonas maltophilia and Bacillus spp. were found in all oocyte/embryo bank LN containers. There was no relationship between the number of samples or the time that each container had been used and the presence of microbiologic contaminants in the LN. At the container's bottom, Acinetobacter lwoffii, Alcaligenes faecalis ssp. faecalis, and Sphingomonas paucimobilis were found., Conclusion(s): Bacteria cross-contamination may not occur in oocyte/embryo banking in either open or closed storage devices. However, microorganisms can survive in LN. The bacteria cross-contamination risk is no greater for open than for closed containers. Storage containers should be cleaned periodically owing to the risk of lost straws or small particles of contaminated material., (Copyright © 2016. Published by Elsevier Inc.)

Various insects require intracellular bacteria that are restricted to specialized cells (bacteriocytes) and are transmitted vertically via the female ovary, but the transmission mechanisms are obscure. We hypothesized that, in the whitefly Bemisia tabaci, where intact bacteriocytes (and not isolated bacteria) are transferred to oocytes, the transmission mechanism would be evident as cellular and molecular differences between the nymph (pre-adult) and adult bacteriocytes. We demonstrate dramatic remodelling of bacteriocytes at the developmental transition from nymph to adulthood. This transition involves the loss of cell-cell adhesion, high division rates to constant cell size and onset of cell mobility, enabling the bacteriocytes to crawl to the ovaries. These changes are accompanied by cytoskeleton reorganization and changes in gene expression: genes functioning in cell-cell adhesion display reduced expression and genes involved in cell division, cell motility and endocytosis/exocytosis have elevated expression in adult bacteriocytes, relative to nymph bacteriocytes. This study demonstrates, for the first time, how developmentally orchestrated remodelling of gene expression and correlated changes in cell behaviour underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends., (© 2016 The Authors.)

The leafhopper Macrosteles laevis, like other plant sap-feeding hemipterans, lives in obligate symbiotic association with microorganisms. The symbionts are harbored in the cytoplasm of large cells termed bacteriocytes, which are integrated into huge organs termed bacteriomes. Morphological and molecular investigations have revealed that in the bacteriomes of M. laevis, two types of bacteriocytes are present which are as follows: bacteriocytes with bacterium Sulcia and bacteriocytes with Nasuia symbiont. We observed that in bacteriocytes with Sulcia, some cells of this bacterium contain numerous cells of the bacterium Arsenophonus. All types of symbionts are transmitted transovarially between generations. In the mature female, the bacteria Nasuia, bacteria Sulcia, and Sulcia with Arsenophonus inside are released from the bacteriocytes and start to assemble around the terminal oocytes. Next, the bacteria enter the cytoplasm of follicular cells surrounding the posterior pole of the oocyte. After passing through the follicular cells, the symbionts enter the space between the oocyte and follicular epithelium, forming a characteristic "symbiont ball."

Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1's role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.

Background: Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results., Objective: The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus., Results: Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes., Conclusions: These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view.

Carsonella ruddii (Gamma Proteobacterium) is an obligate bacterial endosymbiont of psyllids that produces essential amino acids that are lacking in the insect's diet. Accurate estimations of Carsonella populations are important to studies of Carsonella-psyllid interactions and to developing ways to target Carsonella for control of psyllid pests including pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae) and potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae). We used two methods, namely fluorescence in situ hybridization and quantitative polymerase chain reaction (qPCR), to estimate relative abundance of Carsonella in bacteriocytes and whole bodies of psyllids, respectively. Using these two methods, we compared Carsonella populations between female and male insects. Estimations using fluorescence in situ hybridization indicated that Carsonella was more abundant in bacteriocytes of female C. pyricola than in those of males, but Carsonella abundance in bacteriocytes did not differ between sexes of B. cockerelli. Analyses by qPCR using whole-body specimens indicated Carsonella was more abundant in females than in males of both psyllids. Neither fluorescence in situ hybridization nor qPCR indicated that Carsonella populations differed in abundance among adults of different ages (0-3 wk after adult eclosion). Using fluorescence in situ hybridization, Carsonella was observed in ovarioles of newly emerged females and formed an aggregation in the posterior end of mature oocytes. Results of our study indicate that female psyllids harbor greater populations of Carsonella than do males and that sex should be controlled for in studies which require estimations of Carsonella populations., (Published by Oxford University Press on behalf of the Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US.)

While a number of studies have identified host factors that influence endosymbiont titer, little is known concerning environmental influences on titer. Here we examined nutrient impact on maternally transmitted Wolbachia endosymbionts in Drosophila. We demonstrate that Drosophila reared on sucrose- and yeast-enriched diets exhibit increased and reduced Wolbachia titers in oogenesis, respectively. The yeast-induced Wolbachia depletion is mediated in large part by the somatic TOR and insulin signaling pathways. Disrupting TORC1 with the small molecule rapamycin dramatically increases oocyte Wolbachia titer, whereas hyper-activating somatic TORC1 suppresses oocyte titer. Furthermore, genetic ablation of insulin-producing cells located in the Drosophila brain abolished the yeast impact on oocyte titer. Exposure to yeast-enriched diets altered Wolbachia nucleoid morphology in oogenesis. Furthermore, dietary yeast increased somatic Wolbachia titer overall, though not in the central nervous system. These findings highlight the interactions between Wolbachia and germline cells as strongly nutrient-sensitive, and implicate conserved host signaling pathways by which nutrients influence Wolbachia titer.

Arthropod symbionts present tissue tropism that corresponds to the nature of the association and the mode of transmission between host generations. In ticks, however, our knowledge of symbiont tissue tropism and function is limited. Here, we quantified and localized previously described Coxiella-like symbionts in several organs of the tick Rhipicephalus turanicus. Quantitative polymerase chain reaction revealed high densities of Coxiella in the female gonads, and both male and female Malpighian tubules. Using fluorescence in situ hybridization and transmission electron microscopy, we further showed that in the gonads of both Rh. turanicus and Rh. sanguineus, Coxiella does not colonize the primary oocytes but is found later in young and mature oocytes in a specific distribution, suggesting controlled vertical transmission. This method revealed the presence Coxiella in the distal part of the Malpighian tubules, suggesting a possible role in nitrogen metabolism. While testing Rickettsia symbionts, no specific tissue tropism was found, but a slightly higher densities in the tick gut. The low density of Rickettsia in the female ovaries suggests competition between Rickettsia and Coxiella for vertical transmission. The described tissue distribution supports an obligatory role for Coxiella in ticks., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Unlabelled: The endosymbiotic bacteria of the genus Wolbachia are present in most insects and are maternally transmitted through the germline. Moreover, these intracellular bacteria exert antiviral activity against insect RNA viruses, as in Drosophila melanogaster, which could explain the prevalence of Wolbachia bacteria in natural populations. Wolbachia is maternally transmitted in D. melanogaster through a mechanism that involves distribution at the posterior pole of mature oocytes and then incorporation into the pole cells of the embryos. In parallel, maternal transmission of several endogenous retroviruses is well documented in D. melanogaster. Notably, gypsy retrovirus is expressed in permissive follicle cells and transferred to the oocyte and then to the offspring by integrating into their genomes. Here, we show that the presence of Wolbachia wMel reduces the rate of gypsy insertion into the ovo gene. However, the presence of Wolbachia does not modify the expression levels of gypsy RNA and envelope glycoprotein from either permissive or restrictive ovaries. Moreover, Wolbachia affects the pattern of distribution of the retroviral particles and the gypsy envelope protein in permissive follicle cells. Altogether, our results enlarge the knowledge of the antiviral activity of Wolbachia to include reducing the maternal transmission of endogenous retroviruses in D. melanogaster., Importance: Animals have established complex relationships with bacteria and viruses that spread horizontally among individuals or are vertically transmitted, i.e., from parents to offspring. It is well established that members of the genus Wolbachia, maternally inherited symbiotic bacteria present mainly in arthropods, reduce the replication of several RNA viruses transmitted horizontally. Here, we demonstrate for the first time that Wolbachia diminishes the maternal transmission of gypsy, an endogenous retrovirus in Drosophila melanogaster. We hypothesize that gypsy cannot efficiently integrate into the germ cells of offspring during embryonic development in the presence of Wolbachia because both are competitors for localization to the posterior pole of the egg. More generally, it would be of interest to analyze the influence of Wolbachia on vertically transmitted exogenous viruses, such as some arboviruses., (Copyright © 2014 Touret et al.)

Microbial symbionts played a central role in insect evolution. Oreina cacaliae (Schrank, 1785) (Coleoptera: Chrysomelidae) is a rare example of a viviparous insect, able to feed on toxic plants and sequester toxic compounds. In the current study, the microbiota associated with O. cacaliae was characterized using a culture-independent approach, targeting the 16S rRNA bacterial gene. The obtained 16S rRNA gene sequences were analyzed and identified at different taxonomic levels. Wolbachia was the dominant bacterium, both in male and female (100 and 91.9%, respectively) individuals; the detected Wolbachia was described as a new sequence type based on multilocus sequence typing (Wolbachia ST375 Ocac&#95;A&#95;wVdO). After phylogenetic analyses, Wolbachia ST375 Ocac&#95;A&#95;wVdO was attributed to the supergroup A. Immunofluorescence assays and electron microscopy confirmed the presence of Wolbachia within O. cacaliae oocytes, confirming its transovarial transmission in this species. Representatives of six species of Oreina were tested for the presence of Wolbachia through specific polymerase chain reaction, and a dendrogram was generated for these species based on coxI gene sequences. The Wolbachia harbored by different species of Oreina were characterized by multilocus sequence typing. Five out of the six examined Oreina species were positive for Wolbachia, with four of these harboring the same sequence type.

Previously, we found that oocyte-secreted factors (OSFs) secreted by denuded oocytes during in vitro maturation (IVM) enhance subsequent development of bovine somatic cell nuclear transfer (SCNT) embryos. This treatment requires many oocytes during IVM. Hence, the aim of this study was to investigate whether supplementing with recombinant growth differentiation factor-9 (GDF9), one of crucial OFSs, in oocyte maturation medium could improve developmental competence of bovine oocytes and subsequent development of cloned embryos. Cumulus-oocyte complexes (COCs) from antral follicles of bovine ovaries collected from an abattoir were cultured with (SCNT+GDF9 group) or without (SCNT group) 200 ng/mL recombinant human GDF9 in oocyte maturation medium. After 22 h, metaphase II (MII) oocytes were used for SCNT. The presence of 200 ng/mL GDF9 significantly increased oocyte maturation rates, the cleavage rate, and blastocyst formation rates of bovine cloned embryos. The blastocyst total, inner cell mass (ICM) cell numbers, and ratio of ICM:TE were higher, whereas the rate of apoptosis in bovine cloned blastocysts was lower in the SCNT+GDF9 group than in the SCNT group. The histone modifications at various sites were also different between each group. These results suggest that COCs cultured with recombinant GDF9 in oocyte maturation medium improve oocyte developmental competence and subsequent developmental competence of cloned embryo in cattle.

Wolbachia are widespread endosymbiotic bacteria of arthropods and nematodes. Studies on such models suggest that Wolbachia's remarkable aptitude to infect offspring may rely on a re-infection of ovaries from somatic tissues instead of direct cellular segregation between oogonia and oocytes. In the terrestrial isopod Armadillidium vulgare, Wolbachia are vertically transmitted to the host offspring, even though ovary cells are cyclically renewed. Using Fluorescence in situ hybridization (FISH), we showed that the proportion of infected oocytes increased in the course of ovary and oocyte maturation, starting with 31.5% of infected oocytes only. At the end of ovary maturation, this proportion reached 87.6% for the most mature oocytes, which is close to the known transmission rate to offspring. This enrichment can be explained by a secondary acquisition of the bacteria by oocytes (Wolbachia can be seen as last minute passengers) and/or by a preferential selection of oocytes infected with Wolbachia (as priority travellers).

Mastitis is associated with decreased fertility in dairy cows. In the current study, we created an experimental model to simulate short-term mastitis by a single intramammary administration of Gram-negative endotoxin of Escherichia coli origin (G-), or Gram-positive toxin of Staphylococcus aureus origin (G+), to examine the effect of mastitis on oocyte developmental competence. Healthy Holstein cows were synchronized, and follicular fluid (FF) of cows treated with G+ or G- and of uninfected cows (controls) was aspirated from the preovulatory follicles by transvaginal ultrasound procedure. The aspirated FF was used as maturation medium for in vitro embryo production. The distribution of matured oocytes into different cortical granule classes and meiotic stages was affected by G- administration (P<0.05) but not by G+ administration. The proportion of oocytes that cleaved to two- and four-cell stage embryos (44 h postfertilization) was lower in both G+ and G- groups than in controls (P<0.05). Blastocyst formation rate (7-8 days postfertilization) was lower in the G- group (P<0.05) and numerically lower in the G+ group compared with their uninfected counterparts. The total cell number in blastocysts did not differ among groups; however, the apoptotic index was higher in the G+ group (P<0.05), but not in the G- group, relative to controls. Examining mRNA relative abundance in oocytes and early embryos revealed mastitis-induced alterations in PTGS2 (COX2), POU5F1, and HSF1 but not in SLC2A1 (GLUT1) or GDF9. Results indicate a differential disruptive effect of mastitis induced by G- and G+ on oocyte developmental competence in association with alterations in maternal gene expression.

The aim of this paper was to identify endosymbiotic microorganisms living in the body cavity of a Polish population of an aphid, Adelges (Sacchiphantes) viridis, as well as to describe their ultrastructure and mode of transmission between generations. Molecular data (amplification and sequencing of 16S rRNA genes) indicated that endosymbionts of A. (S.) viridis are Betaproteobacteria of the species "Candidatus Vallotia virida". Endosymbiotic bacteria are rod-shaped and localized in the cytoplasm of specific cells, termed bacteriocytes, of host insects. Endosymbionts sharing the same bacteriocytes differ in the density of their cytoplasm. There are two morphotypes of endosymbiotic bacteria: with electron-dense cytoplasm and electron-translucent cytoplasm. Since only bacteria containing electron-dense cytoplasm were observed in the binary fusion stage, differences in density of the cytoplasm are probably due to changes in the cytoskeleton of bacteria during division. Endosymbionts of A. (S.) viridis are transovarially (i.e. via oocytes) transmitted from the mother to the offspring., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the germinal vesicle stage). The disruption was associated with elevation of SCC rather than bacterial type. The results may provide a partial explanation for the low fertility of cows that have contracted mastitic pathogens before insemination., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Recently we reported the presence of bacteria within follicular fluid. Previous studies have reported that DNA fragmentation in human spermatozoa after in vivo or in vitro incubation with bacteria results in early embryo demise and a reduced rate of ongoing pregnancy, but the effect of bacteria on oocytes is unknown. This study examined the DNA within mouse oocytes after 12 hours' incubation within human follicular fluids (n=5), which were collected from women undergoing in vitro fertilization (IVF) treatment. Each follicular fluid sample was cultured to detect the presence of bacteria. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was used to label DNA fragmentation in ovulated, non-fertilized mouse oocytes following in vitro incubation in human follicular fluid. The bacteria Streptococcus anginosus and Peptoniphilus spp., Lactobacillus gasseri (low-dose), L. gasseri (high-dose), Enterococcus faecalis, or Propionibacterium acnes were detected within the follicular fluids. The most severe DNA fragmentation was observed in oocytes incubated in the follicular fluids containing P. acnes or L. gasseri (high-dose). No DNA fragmentation was observed in the mouse oocytes incubated in the follicular fluid containing low-dose L. gasseri or E. faecalis. Low human oocyte fertilization rates (<29%) were associated with extensive fragmentation in mouse oocytes (80-100%). Bacteria colonizing human follicular fluid in vivo may cause DNA fragmentation in mouse oocytes following 12h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: Spiroplasma is a diverse bacterial clade that includes many vertically transmitted insect endosymbionts, including Spiroplasma poulsonii, a natural endosymbiont of Drosophila melanogaster. These bacteria persist in the hemolymph of their adult host and exhibit efficient vertical transmission from mother to offspring. In this study, we analyzed the mechanism that underlies their vertical transmission, and here we provide strong evidence that these bacteria use the yolk uptake machinery to colonize the germ line. We show that Spiroplasma reaches the oocyte by passing through the intercellular space surrounding the ovarian follicle cells and is then endocytosed into oocytes within yolk granules during the vitellogenic stages of oogenesis. Mutations that disrupt yolk uptake by oocytes inhibit vertical Spiroplasma transmission and lead to an accumulation of these bacteria outside the oocyte. Impairment of yolk secretion by the fat body results in Spiroplasma not reaching the oocyte and a severe reduction of vertical transmission. We propose a model in which Spiroplasma first interacts with yolk in the hemolymph to gain access to the oocyte and then uses the yolk receptor, Yolkless, to be endocytosed into the oocyte. Cooption of the yolk uptake machinery is a powerful strategy for endosymbionts to target the germ line and achieve vertical transmission. This mechanism may apply to other endosymbionts and provides a possible explanation for endosymbiont host specificity., Importance: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. Studies have shown that many facultative endosymbionts, including Spiroplasma, confer protection against different classes of parasites on their hosts and therefore are attractive tools for the control of vector-borne diseases. The ability to be efficiently transmitted from females to their offspring is the key feature shaping associations between insects and their inherited endosymbionts, but to date, little is known about the mechanisms involved. In oviparous animals, yolk accumulates in developing eggs and serves to meet the nutritional demands of embryonic development. Here we show that Spiroplasma coopts the yolk transport and uptake machinery to colonize the germ line and ensure efficient vertical transmission. The uptake of yolk is a female germ line-specific feature and therefore an attractive target for cooption by endosymbionts that need to maintain high-fidelity maternal transmission.

Microsporidia are unicellular fungi that are obligate endoparasites. Although nematodes are one of the most abundant and diverse animal groups, the only confirmed report of microsporidian infection was that of the "nematode killer" (Nematocida parisii). N. parisii was isolated from a wild Caenorhabditis sp. and causes an acute and lethal intestinal infection in a lab strain of Caenorhabditis elegans. We set out to characterize a microsporidian infection in a wild nematode to determine whether the infection pattern of N. parisii in the lab is typical of microsporidian infections in nematodes. We describe a novel microsporidian species named Sporanauta perivermis (marine spore of roundworms) and characterize its infection in its natural host, the free-living marine nematode Odontophora rectangula. S. perivermis is not closely related to N. parisii and differs strikingly in all aspects of infection. Examination by transmission electron microscopy (TEM) revealed that the infection was localized in the hypodermal and muscle tissues only and did not involve the intestines. Fluorescent in situ hybridization (FISH) confirmed infection in the muscle and hypodermis, and surprisingly, it also revealed that the parasite infects O. rectangula eggs, suggesting a vertical mode of transmission. Our observations highlight the importance of studying parasites in their natural hosts and indicate that not all nematode-infecting microsporidia are "nematode killers"; instead, microsporidiosis can be more versatile and chronic in the wild.

Nomuraea rileyi, a fungus pathogenic to insects, has been widely used for biological control of agricultural pests in Brazil. This study investigates the effects of N. rileyi, isolates Nr 138, Nr 151, and Nr 177, to eggs, larvae, and engorged females of Rhipicephalus microplus tick. Specimens were immersed in 1 ml of conidial suspension for 3 min, whereas the control group was immersed in 0.01% Tween 80 water solution. The isolate Nr 138 controlled 67.37% of ticks when the highest conidial concentration was used, 10(8) conidia ml(-1). The isolate Nr 177 significantly reduced the percentage of hatch of larvae from eggs treated with 10(8) conidia ml(-1). Conversely, the isolate Nr 151 was not virulent to eggs, larvae, or adults. Variability in virulence was observed among the N. rileyi isolates investigated in the current study-Nr 138 was more virulent to engorged females, while Nr 177 was more virulent to unfed larvae. Although N. rileyi proved to be virulent to several stages of R. microplus, the results obtained in this study indicate that N. rileyi does not appear to be a remarkable biological control agent for R. microplus.

Objective: To report a washing procedure, to be performed as frozen specimens are taken out of cryobanks, to minimize the risk of hypothetical culture contamination during thawing., Design: Basic research., Setting: Private assisted reproduction center., Intervention(s): Two batches of liquid nitrogen (LN(2)) were experimentally contaminated, one with bacteria (Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophilia) and the other with fungi (Aspergillus niger). Two hundred thirty-two of the most common human gamete/embryo vitrification carriers (Cryotop, Cryoleaf, Cryopette) were immersed in the contaminated LN(2) (117 in the bacteria and 25 in the fungi-contaminated LN(2)). The carriers were tested microbiologically, one group without washing (control) and the other after three subsequent washings in certified ultraviolet sterile liquid nitrogen (SLN(2)). The carriers were randomly allocated to the "three-wash procedure" (three-wash group, 142 carriers) or "no-wash" (control group, 90 carriers) using a specific software tool., Mean Outcome Measure(s): Assessment of microorganism growth., Result(s): In the no-wash control group, 78.6% of the carriers were contaminated by the bacteria and 100% by the fungi. No carriers were found to be contaminated, either by bacteria or fungi, after the three-wash procedure., Conclusion(s): The three-wash procedure with SLN(2) produced an efficient decontamination of carriers in extreme experimental conditions. For this reason, this procedure could be routinely performed in IVF laboratories for safe thawing of human specimens that are cryostored in nonhermetical cryocontainers, particularly in the case of open or single-straw closed vitrification systems., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Wolbachia bacteria are ubiquitous intracellular bacteria of arthropods. Often considered reproductive parasites, they can benefit certain host species. We describe a new Wolbachia strain from Leptopilina victoriae, a Drosophila wasp. The strain is closely related to Wolbachia from Culex sp. Located to the posterior poles of oocytes, it manipulates its host's reproduction by inducing a male development type of cytoplasmic incompatibility. We also report its diverse effects on the wasp's life history traits.

The whitefly Bemisia tabaci is a cosmopolitan insect pest that harbors Portiera aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative secondary symbionts. Secondary symbionts in B. tabaci are generally associated with the bacteriome, ensuring their vertical transmission; however, Rickettsia is an exception and occupies most of the body cavity, except the bacteriome. The mode of Rickettsia transfer between generations and its subcellular localization in insect organs have not been investigated. Using electron and fluorescence microscopy, we show that Rickettsia infects the digestive, salivary, and reproductive organs of the insect; however, it was not observed in the bacteriome. Rickettsia invades the oocytes during early developmental stages and resides in follicular cells and cytoplasm; it is mostly excluded when the egg matures; however, some bacterial cells remain in the egg, ensuring their transfer to subsequent generations. Rickettsia was localized to testicles and the spermatheca, suggesting a horizontal transfer between males and females during mating. The bacterium was further observed at large amounts in midgut cells, concentrating in vacuole-like structures, and was located in the hemolymph, specifically at exceptionally large amounts around bacteriocytes and in fat bodies. Organs further infected by Rickettsia included the primary salivary glands and stylets, sites of possible secretion of the bacterium outside the whitefly body. The close association between Rickettsia and the B. tabaci digestive system might be important for digestive purposes. The vertical transmission of Rickettsia to subsequent generations occurs via the oocyte and not, like other secondary symbionts, the bacteriome.

Background: The γ-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis., Methodology/principal Findings: Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.8 ± 0.8 × 10(1) and 1.1 ± 0.03 × 10(3) CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42% of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50% of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then to the oocytes, but the pathogen was not recovered from the subsequently-hatched larvae., Conclusions/significance: This study demonstrates that D. variabilis can be efficiently colonized with F. tularensis using artificial methods. The persistence of F. tularensis in D. variabilis suggests that this tick species may be involved in the maintenance of enzootic foci of tularemia in the central United States.

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.

Background: Considering the fact that the dog tick, Rhipicephalus sanguineus, has a great potential to become the vector of Brazilian Spotted Fever (BSF) for humans, the present study aimed to describe the distribution of the bacterium Rickettsia rickettsii, the etiological agent of BSF, in different regions of the ovaries of R. sanguineus using histological techniques. The ovaries were obtained from positive females confirmed by the hemolymph test and fed in the nymph stage on guinea pigs inoculated with R. rickettsii., Results: The results showed a general distribution of R. rickettsii in the ovary cells, being found in oocytes in all stages of development (I, II, III, IV and V) most commonly in the periphery of the oocyte and also in the cytoplasm of pedicel cells., Conclusions: The histological analysis of the ovaries of R. sanguineus infected females confirmed the presence of the bacterium, indicating that the infection can interfere negatively in the process of reproduction of the ticks, once alterations were detected both in the shape and cell structure of the oocytes which contained bacteria.

The Xenopus laevis oocyte has been the workhorse for the investigation of ion transport proteins. These large cells have spawned a multitude of novel techniques that are unfathomable in mammalian cells, yet the fickleness of the oocyte has driven many researchers to use other membrane protein expression systems. Here, we show that some colonies of Xenopus laevis are infected with three multi-drug-resistant bacteria: Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Oocytes extracted from infected frogs quickly (3-4 d) develop multiple black foci on the animal pole, similar to microinjection scars, which render the extracted eggs useless for electrical recordings. Although multi-drug resistant, the bacteria were susceptible to amikacin and ciprofloxacin in growth assays. Supplementing the oocyte storage media with these two antibiotics prevented the appearance of the black foci and afforded oocytes suitable for whole-cell recordings. Given that P. fluorescens associated with X. laevis has become rapidly drug resistant, it is imperative that researchers store the extracted oocytes in the antibiotic cocktail and not treat the animals harboring the multi-drug-resistant bacteria.

Cryptosporidiosis is one of the most frequent causes of diarrhea in Haiti. Transmission in children less than five years-old, HIV-infected individuals, and people living in low socio-economic conditions is frequently due to consumption of water or food contaminated by Cryptosporidium oocysts. This study examined the circulation of Cryptosporidium oocysts in surface waters and in public water supplies in the district of Port-au-Prince. Data were gathered from December 2000 to June 2002 in 37 sites. In the district of Port-au-Prince, 24/37 samples of water collected (65%) were contaminated by Cryptosporidium oocysts and 10/11 (91%) of those collected in reservoirs used by people living in peripheral areas. The rate of contamination was 7/13 (54%) in water from public standpipes provided by the public company of water distribution. All surface water (4/4) collected was highly contaminated., (Copyright © 2011 Taylor & Francis Group, LLC)

The aim of this study was to verify the effects of probiotic Lactobacillus rhamnosus on zebrafish oocyte maturation using FPA (focal plane array) FTIR imaging together with specific biochemical assays (SDS-PAGE, real-time PCR and enzymatic assay). Oocyte growth is prevalently due to a vitellogenic process which consists of the hepatic synthesis of vitellogenin and its selective uptake during maturation. The administration of L. rhamnosus IMC 501 for 10 days induced chemical changes to oocyte composition, promoting the maturation process. Some interesting biochemical features, linked to protein secondary structure (amide I band) and to phospholipidic and glucidic patterns, were detailed by vibrational analysis. The spectroscopic results were supported by the early increase of the lysosomal enzyme involved in the final oocyte maturation, the cathepsin L. This enzyme increases during follicle maturation, with the highest levels in class IV oocytes. In treated females, class III oocytes showed higher cathepsin L gene expression and enzymatic activity, with levels comparable to class IV oocytes isolated from controls; this can be related to the proteolytic cleavage of the higher molecular mass yolk protein components, as evidenced by SDS-PAGE.

Cryopreservation of sperm, embryos and, more recently, oocytes plays an important and increasing role in assisted reproduction, due to improvements of old, and introduction of new technologies. In parallel, concerns are increasing about the technical and biological safety of these procedures. However, published data regarding the confirmed or theoretical hazards of these procedures are sparse and sometimes contradictory. The purpose of this review will summarize data and opinions about one of the most disputed risks, the potential hazard of contamination and disease transmission through cryopreservation. Special attention is concentrated on the weak points of the technology including open vitrification systems, sterilization of liquid nitrogen and safety of commonly used storage tanks including straws and cryovials. Suggestions are also made for practical measures to avoid these dangers while preserving the benefits and perspectives of new cryopreservation technologies.

Studies of the biochemistry of Listeria monocytogenes virulence protein ActA have typically focused on the behavior of bacteria in complex systems or on the characterization of the protein after expression and purification. Although prior in vivo work has proposed that ActA forms dimers on the surface of L. monocytogenes, dimerization has not been demonstrated in vitro, and little consideration has been given to the surface environment where ActA performs its pivotal role in bacterial actin-based motility. We have synthesized and characterized an ActA dimer and provide evidence that the two ActA molecules do not interact with each other even when tethered together. However, we also demonstrate that artificial dimers provide superior activation of actin nucleation by the Arp2/3 complex compared with monomers and that increased activation of the Arp2/3 complex by dimers may be a general property of Arp2/3 activators. It appears that the close packing ( approximately 19 nm) of ActA molecules on the surface of L. monocytogenes is so dense that the kinetics of actin nucleation mimic that of synthetic ActA dimers. We also present observations indicating that ActA is a natively unfolded protein, largely random coil that is responsible for many of the unique physical properties of ActA including its extended structure, aberrant mobility during SDS-PAGE, and ability to resist irreversible denaturation upon heating.

Wolbachia are gram-negative bacteria that are widespread in nature, carried by the majority of insect species as well as some mites, crustaceans, and filarial nematodes. Wolbachia can range from parasitic to symbiotic, depending upon the interaction with the host species. The success of Wolbachia is attributed to efficient maternal transmission and manipulations of host reproduction that favor infected females, such as sperm-egg cytoplasmic incompatibility (CI). Much remains unknown about the mechanistic basis for Wolbachia-host interactions. Here we summarize the current understanding of Wolbachia interaction with insect hosts, with a focus on Drosophila. The areas of discussion include Wolbachia transmission in oogenesis, Wolbachia distribution in spermatogenesis, induction and rescue of the CI phenotype, Wolbachia genomics, and Wolbachia-membrane interactions.

Wolbachia are among the most widespread intracellular bacteria, carried by thousands of metazoan species. The success of Wolbachia is due to efficient vertical transmission by the host maternal germline. Some Wolbachia strains concentrate at the posterior of host oocytes, which promotes Wolbachia incorporation into posterior germ cells during embryogenesis. The molecular basis for this localization strategy is unknown. Here we report that the wMel Wolbachia strain relies upon a two-step mechanism for its posterior localization in oogenesis. The microtubule motor protein kinesin-1 transports wMel toward the oocyte posterior, then pole plasm mediates wMel anchorage to the posterior cortex. Trans-infection tests demonstrate that factors intrinsic to Wolbachia are responsible for directing posterior Wolbachia localization in oogenesis. These findings indicate that Wolbachia can direct the cellular machinery of host oocytes to promote germline-based bacterial transmission. This study also suggests parallels between Wolbachia localization mechanisms and those used by other intracellular pathogens.