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Three novel bacterial strains, FE4 T , FE10 T , and LA51 T , which are phylogenetically affiliated to the genera Pseudoalteromonas, Vibrio, or Marinobacter, respectively, isolated from fertilized eggs and juveniles of sea cucumber Apostichopus japonicus were characterized by a genome-based taxonomical approach including multilocus sequence analysis (MLSA) combined with classical phenotypic and chemotaxonomic characterizations. A molecular network reconstructed on the basis of nucleotide sequences of four phylogenetic maker protein genes revealed that the strains FE4 T , FE10 T , and LA51 T were closely related to Pseudoalteromonas shioyasakiensis, Vibrio lentus, and Marinobacter similis, respectively. Average nucleotide identity (ANI) comparisons against phylogenetically related species to FE4 T , FE10 T , and LA51 T demonstrated that each newly described strain could not be identified as any previously described species within each genus showing < 95% ANI: 91.3% of FE4 T against P. shioyasakiensis JCM 18891  T , 92.6% of FE10 T against "V. bathopelagicus" Sal10, and 92.6% of LA51 T against M. similis A3d10 T , in maximum, respectively. Here, we show molecular phylogenetic, genomic, phenotypic, and chemotaxonomic features of the newly described species FE4 T , FE10 T , and LA51 T . We also propose Pseudoalteromonas apostichopi sp. nov. with FE4 T (JCM 36173  T  = LMG 33143  T ) as the type strain, Vibrio apostichopi sp. nov. with FE10 T (JCM 36174  T  = LMG 33144  T ) as the type strain, and Marinobacter apostichopi sp. nov. with LA51 T (JCM 36175  T  = LMG 33145  T ) as the type strain., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dengue fever is one of the most severe viral diseases transmitted by Aedes mosquitoes, with traditional approaches of disease control proving insufficient to prevent significant disease burden. Release of Wolbachia-transinfected mosquitoes offers a promising alternative control methodologies; Wolbachia-transinfected female Aedes aegypti demonstrate reduced dengue virus transmission, whilst Wolbachia-transinfected males cause zygotic lethality when crossed with uninfected females, providing a method for suppressing mosquito populations. Although highly promising, the delicate nature of population control strategies and differences between local species populations means that controlled releases of Wolbachia-transinfected mosquitoes cannot be performed without extensive testing on specific local Ae. aegypti populations. In order to investigate the potential for using Wolbachia to suppress local Ae. aegypti populations in Taiwan, we performed lab-based and semi-field fitness trials. We first transinfected the Wolbachia strain wAlbB into a local Ae. aegypti population (wAlbB-Tw) and found no significant changes in lifespan, fecundity and fertility when compared to controls. In the laboratory, we found that as the proportion of released male mosquitoes carrying Wolbachia was increased, population suppression could reach up to 100%. Equivalent experiments in semi-field experiments found suppression rates of up to 70%. The release of different ratios of wAlbB-Tw males in the semi-field system provided an estimate of the optimal size of male releases. Our results indicate that wAlbB-Tw has significant potential for use in vector control strategies aimed at Ae. aegypti population suppression in Taiwan. Open field release trials are now necessary to confirm that wAlbB-Tw mediated suppression is feasible in natural environments., Competing Interests: The authors have declared that no competing interests exist.

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.)

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ + B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ + B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Generally speaking, fish intestinal microbiota is easily affected by food or water environment, and it may be dynamically changed along with body growth. However, it remains unclear whether fish gut microbiota can be affected under any conditions. In the present study, we focused on cultured larval turbot (Scophthalmus maximus) and tracked its artificial breeding process from eggs to larvae in two farms located in different regions of China. Through continuous sampling, we analyzed and compared characteristics of intestinal microbiota in turbot larvae and its correlation with the bacteria in water and food at different developmental stages. The results showed that there was a steady group of microbiota in larval gut, and the highest relative abundance of strain was same between the two farms. This microbiota was established soon after hatching of fertilized eggs. Particularly, the structure of this microbiota was nearly not changeable afterward 3-4 months of development. The bacteria carried by fertilized eggs might play an important role during the formation of this microbiota. In conclusion, our findings suggested that there was a core microbiota represented by Lactococcus sp. in gut of artificially bred turbot larvae. The relative proportion of such strain in gut was higher than 30% at the initial stage of turbot life.

This experiment was conducted to investigate the effects of manganese (Mn) and Bacillus subtilis (BS) on the production performance, egg quality, antioxidant capacity, and gut microbiota of breeding geese during laying period. A total of 120 forty-six-week-old breeding geese (Wulong) were randomly assigned to 1 of 6 treatment diets formulated to supply 10, 20, and 30 mg/kg Mn with 5 × 10 9  CFU/kg or 2.5 × 10 9  CFU/kg BS for a 10-wk trial. Results showed that dietary supplementation with 20 and 30 mg/kg Mn could decrease the daily feed intake (DFI) of geese. Moreover, 30 mg/kg Mn significantly increased the laying rate. Besides, although Mn addition had no obvious effect on egg quality, 5 × 10 9  CFU/kg BS was found to elevate the hatching egg hatching rate and eggshell thickness. For the serum hormones, 30 mg/kg Mn promoted estradiol secretion, while 5 × 10 9  CFU/kg BS increased the level of follicle-stimulating hormone. Furthermore, 20 and 30 mg/kg Mn and 5 × 10 9  CFU/kg BS significantly enhanced the total antioxidant capacity by increasing the activity of total superoxide dismutases or decreasing the content of malondialdehyde. Dietary supplementation with 5 × 10 9  CFU/kg BS also increased the intestinal villus height and upregulated the abundance of Fusobacteria, Fusobacteriaceae, Fusobacterium, and Faecalibacterium in cecal content. In addition, 20 and 30 mg/kg Mn elevated the levels of Bacteroidetes, Bacteroidaceae, Bacteroides, and Ruminococcaceae but decreased Streptococcaceae. Importantly, an interaction effect was observed between Mn and BS on the DFI, egg mass, average egg size, and the abundance of Bacteroides as well as Faecalibacterium. In conclusion, dietary inclusion of Mn and BS could improve the production performance, egg quality, antioxidant capacity, intestinal structure, as well as gut microbiota. Supplementation of 30 mg/kg Mn and 5.0 × 10 9  CFU/kg BS provided the optimal effect., (Copyright © 2020. Published by Elsevier Inc.)

The aim of this study was to evaluate the efficacy of sanitizing fertile eggs with clove essential oil as an alternative to paraformaldehyde; effects on the reduction in eggshell microbial count, incubation yield, and neonatal chick quality were measured. A total of 1,460 brown fertile eggs with a mean weight of 58.64 ± 0.49 g (from 37-wk-old CPK [Pesadão Vermelho] breeder hens) were collected under aseptic conditions and randomly distributed into 4 treatments (nonsanitized and sanitized with grain alcohol, clove essential oil, and paraformaldehyde) before incubation. The count of total aerobic mesophilic bacteria was significantly lower after spraying with clove essential oil (2.30 ± 0.24 log 10  CFU/mL) than on nonsanitized eggs (3.49 ± 0.34 log 10  CFU/mL) or on eggs sprayed with grain alcohol (3.09 ± 0.14 log 10  CFU/mL) but did not differ significantly from the count in the paraformaldehyde group (2.23 ± 0.29 log 10 CFU/mL). The hatchability of fertile eggs differed significantly between the studied treatments. The mean values for the eggs treated with clove essential oil (84.69 ± 1.65%) and paraformaldehyde (81.87 ± 3.92%) were statistically similar but were higher than the negative control (74.03 ± 3.58%) and grain alcohol (73.59 ± 2.87%) values. In the Pasgar© score assessment, it was determined that the clove essential oil (9.21 ± 0.89%) had a superior effect on the physical quality of the chicks compared with the effects of the other treatments. Clove essential oil is effective and safe for eggs intended for incubation. Its use as an alternative to paraformaldehyde in the sanitation of fertile eggs is strongly recommended., (Copyright © 2020. Published by Elsevier Inc.)

Previous studies have suggested the use of probiotics, as alternative to antibiotics, to enhance broiler performance. The administration of probiotics in feed has been widely explored; however, few studies have evaluated the in ovo inoculation of probiotics. Therefore, the objective was to evaluate the impact of in ovo inoculation of different concentrations of GalliPro Hatch (GH), an Enterococcus faecium-based probiotic, on hatchability, live performance, and gastrointestinal parameters. Ross x Ross 708 fertile eggs were incubated, and on day 18, injected with the following treatments: 1) 50 μL of Marek's vaccine (MV), 2) MV and 1.4 × 10 5  cfu GH/50 μL, 3) MV and 1.4 × 10 6  cfu GH/50 μL, 4) MV and 1.4 × 10 7  cfu GH/50 μL. On the day of hatch, chicks were weighed, feather sexed, and hatch residue was analyzed. Male birds (640) were randomly assigned to 40 floor pens. On day 0, 7, 14, and 21 of the grow-out phase, performance data were collected. One bird from each pen was used to obtain yolk weight and intestinal segment weight and length. Hatchability was not impacted by any GH treatment (P = 0.58). On day 0, yolk weight was lower for all treatments than for MV alone. On day 0 to 7, feed intake was lower for 10 5 and 10 7  GH; the feed conversion ratio (FCR) was lower for all treatments than for MV alone (P = 0.05; P = 0.01, respectively). From day 14 to 21, the 10 7  GH treatment had higher BW gain (P = 0.05). For day 0 to 21, 10 7  GH had a lower FCR than MV alone (P = 0.03). On day 0, all GH treatments resulted in heavier tissues and longer jejunum, ileum, and ceca lengths than MV alone (P < 0.05). Spleen weight was higher for 10 5 and 10 7  GH than for MV alone. In conclusion, GH does not impact hatchability, and some concentrations improved live performance through the first 21 d of the grow-out phase. These improvements could result from the increased yolk absorption and improved intestinal and spleen morphology seen in this study., (Copyright © 2020. Published by Elsevier Inc.)

Caretta caretta is threatened by many dangers in the Mediterranean basin, but most are human-related. The purposes of this research were: (i) to investigate microflora in samples from six loggerhead sea turtle nests located on the Sicilian coast and (ii) to understand microbial diversity associated with nests, with particular attention to bacteria and fungi involved in failed hatchings. During the 2016 and 2018 summers, 456 eggs and seven dead hatchling from six nests were collected. We performed bacteriological and mycological analyses on 88 egg samples and seven dead hatchlings, allowing us to isolate: Fusarium spp. (80.6%), Aeromonas hydrophila (55.6%), Aspergillus spp. (27.2%) and Citrobacter freundii (9%). Two Fusarium species were identified by microscopy and were confirmed by PCR and internal transcribed spacer sequencing. Statistical analyses showed significant differences between nests and the presence/absence of microflora, whereas no significant differences were observed between eggs and nests. This is the first report that catalogues microflora from C . caretta nests/eggs in the Mediterranean Sea and provides key information on potential pathogens that may affect hatching success. Moreover, our results suggest the need for wider investigations over extensive areas to identify other microflora, and to better understand hatching failures and mortality related to microbial contamination in this important turtle species., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Francisella noatunensis subsp. orientalis (Fno) has been reported as an important bacterial pathogen causing significant mortality (30-95%) in farmed tilapia in broad geographic areas. However, we found that there was a proportion of broodfish in our laboratory that appeared to be healthy but which tested positive for Fno. We therefore hypothesized that Fno might be able to be transmitted from subclinically infected tilapia mouthbrooders to their offspring through the current practice of fry production in tilapia hatcheries. To prove this, experimentally infected hybrid red tilapia broodstock were mated and their offspring were examined for the presence of Fno. In this study, three pairs of infected broodfish were mated for natural spawning and fertilized eggs from each couple were then collected from the female mouths for artificial incubation. The newly hatched larvae were cultured for 30 days and sample collection was performed at different developmental stages i.e. yolk-sac larvae, 5 and 30-day old fry. The results showed that the ovary and testis of all 3 pairs of the broodstock, as well as their fertilized eggs and offspring were Fno positive by Fno-specific PCR and in situ DNA hybridization. In summary, this study revealed that with the current practice in tilapia hatcheries, Fno might be able to transmit from subclinically infected tilapia mouthbrooders to their offspring. Therefore, using Fno-free broodfish in tilapia hatcheries should be considered in order to produce Fno-free tilapia fry., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

One of the most widely reared fish in the Mediterranean Sea is Sparus aurata . The succession of S. aurata whole-body microbiota in fertilized eggs, five, 15, 21 and 71 days post hatch (dph) larvae and the contribution of the rearing water and the provided feed (rotifers, Artemia sp. and commercial diet) to the host's microbiota was investigated by 454 pyrosequencing of the 16S rRNA gene diversity. In total, 1917 bacterial operational taxonomic units (OTUs) were found in all samples. On average, between 93 ± 2.1 and 366 ± 9.2 bacterial OTUs per sample were found, with most of them belonging to Proteobacteria and Bacteroidetes. Ten OTUs were shared between all S. aurata stages and were also detected in the rearing water or diet. The highest OTU richness occurred at the egg stage and the lowest at the yolk sac stage (5 dph). The rearing water and diet microbial communities contributed in S. aurata microbiota without overlaps in their microbial composition and structure. The commercial diet showed higher contribution to the S. aurata microbiota than the rearing water. After stage D71 the observed microbiota showed similarities with that of adult S. aurata as indicated by the increased number of OTUs associated with γ-Proteobacteria and Firmicutes., Competing Interests: The authors declare no conflict of interest.

Based on multiple locus sequence typing, we previously found that DST659 and DST693 were dominant genotypes of Candida albicans among the bloodstream isolates at Chang-Gung Memorial Hospital at Linkou. Biofilm-forming activity, which is critical for C. albicans virulence, probably contributed to the dominance of antifungal sensitive isolates in hospital. Both in vitro membrane weighting and in vivo zebrafish egg infection assays were used to evaluate the biofilm-forming activity of DST659 and DST693 genotypes. Medical records of the patients infected by these two genotypes were retrospectively reviewed. High biofilm-forming activity of DST659 isolates was demonstrated in vitro and further proved with the zebrafish egg infection model, which showed a positive correlation between the biofilm-forming extent on chorion and the in vitro biofilm activity. Moreover, significantly less embryos survived when infected with DST659 isolates than those with DST693 (1.25% vs. 11.43%), and the high-biofilm subset of DST659 showed a greater reduction in survival of embryos at 48 h post-infection than the low-biofilm subset (0 vs. 1.92%). Patients infected with DST659 seemed to survive slightly worse than those infected with DST693, although the difference was insignificant. It is noteworthy that DST659-infected patients were associated with a higher incidence in renal insufficiency as compared to those with DST693, the low biofilm genotype. We suggest that a strong biofilm activity of DST659 contributed to a high mortality rate in zebrafish hosts and poor renal function in patients, as well as gaining the dominance in the northern Taiwan.

Wolbachia is a group of intracellular bacteria that infect a wide range of arthropods including the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. This insect is the vector of Candidatus Liberibacter asiaticus (CLas), the causal pathogen of Huanglongbing or citrus greening disease. Here, we investigated the localization pattern and infection dynamics of Wolbachia in different developmental stages of ACP. Results revealed that all developmental stages of ACP including egg, 1st-5th instar nymphs, and adults of both gender were infected with Wolbachia. FISH visualization of an ACP egg showed that Wolbachia moved from the egg stalk of newly laid eggs to a randomly distributed pattern throughout the egg prior to hatching. The infection rate varied between nymphal instars. The titers of Wolbachia in fourth and fifth instar nymphs were significantly higher than those in the first and second instar nymphs. Wolbachia were scattered in all nymphal stages, but with highest intensity in the U-shaped bacteriome located in the abdomen of the nymph. Wolbachia was confined to two symmetrical organizations in the abdomen of newly emerged female and male adults. The potential mechanisms of Wolbachia infection dynamics are discussed., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Fungal root endophytes, including the common group of dark septate endophytes (DSEs), represent different taxonomic groups and potentially diverse life strategies. In this study, we investigated two unidentified helotialean lineages found previously in a study of DSE fungi of semiarid grasslands, from several other sites, and collected recently from a pezizalean truffle ascoma and eggs of the cereal cyst nematode Heterodera filipjevi. The taxonomic positions and phylogenetic relationships of 21 isolates with different hosts and geographic origins were studied in detail. Four loci, namely, nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS]), partial 28S nuc rDNA (28S), partial 18S nuc rDNA (18S), and partial RNA polymerase II second-largest subunit (RPB2), were amplified and sequenced for molecular phylogenetic analyses. Analyses of similar ITS sequences from public databases revealed two globally distributed lineages detected in several biomes from different geographic regions. The host interaction of isolates from nematodes was examined using in vitro bioassays, which revealed that the fungi could penetrate nematode cysts and colonize eggs of H. filipjevi, confirming observations from field-collected samples. This is the first report of a DSE, and we are not aware of other helotialean fungal species colonizing the eggs of a plant-parasitic nematode. Neither conidiomata and conidia nor ascomata formation was detected in any of the isolates. Based on molecular phylogenetic analyses, these isolates represent a distinct lineage within the Helotiales in the Hyaloscyphaceae. For this lineage, we propose here the new genus Polyphilus represented by two new species, P. sieberi and P. frankenii.

In Focus: Mushegian, A. A., Walser, J. -C., Sullam, K. E., & Ebert, D. (2018). The microbiota of diapause: How host-microbe associations are formed after dormancy in an aquatic crustacean. Journal of Animal Ecology, 87, 400-413. https://doi.org/10.1111/1365-2656.12709. All animals are colonized by micro-organisms, most of which are benign or beneficial. Where do these micro-organisms come from? Theory predicts that micro-organisms which are transmitted vertically from parent to offspring are especially likely to be beneficial to the host, while horizontally acquired micro-organisms are opportunistic and more variable in their impact on host performance. In this issue, Mushegian et al. () investigate the source of bacteria that are required for the growth and development of Daphnia water fleas to reproductive adults. They find that, although vertically transmitted bacteria can occur in the capsule enclosing the Daphnia eggs, the micro-organisms that promote Daphnia performance are associated with the external surface of the capsule and are of likely environmental origin. This mode of transmission may be adaptive for Daphnia because, linked to the longevity and capacity for long-distance dispersal of these eggs, the environmental circumstances encountered by parent and offspring may be different; with the implication, the parental micro-organisms may not be optimal for the offspring. This study demonstrates that, although some animals require symbioses with specific coevolved, vertically transmitted microbial symbionts, other animals have evolved dependence on taxonomically variable micro-organisms of environmental origin., (© 2018 The Author. Journal of Animal Ecology © 2018 British Ecological Society.)

Symbionts are associated with many insects and play several multifunctional roles in insect-microorganism mutualistic relationships. The trichlorphon-degrading symbiont Citrobacter freundii (CF-BD) of the oriental fruit fly Bactrocera dorsalis was recently discovered; however, its intraspecies transmission pathway among flies remains unknown. Here, we use fluorescence in situ hybridization (FISH), PCR detection, and a series of ingenious experiments to reveal that CF-BD was aggregated in rectal pads associated with the female ovipositor, and the CF-BD symbiont was vertically transmitted via egg surface contamination. Although CF-BD was not detected in ovaries, it was found in deposited eggs. In addition, CF-BD was readily acquired horizontally between larvae or adults via oral uptake, although it was not transferred via mating behavior. Surface sterilization of eggs had a negative effect on the insects, which exhibited a lower body weight and a sharp decrease in fecundity, suggesting important biological roles of CF-BD in the fitness of the host insects. Our findings may also help to explain the high pesticide resistance levels of B. dorsalis. Furthermore, identifying a clear transmission pathway of this organophosphorus-degrading symbiont will be useful for pesticide resistance management and future pest control technologies.

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol., (Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture.

Anaplasma marginale is an obligate intracellular pathogen that infects the erythrocytes of calves, causing bovine anaplasmosis. This rickettsia is biologically transmitted by several species of ticks. In tropical and subtropical regions of the world, Rhipicephalus microplus is the main vector. Due to their mobility and longevity, the adult males play an important role in the transmission of A. marginale to calves. Some studies have demonstrated that A. marginale can be intrastadially and interstadially transmitted in R. microplus, but the transovarial transmission has not been demonstrated so far. In the present study, we investigated the effects of low temperature on both the A. marginale migration from infected females to their offspring and reproductive parameters of the tick R. microplus. The larvae of R. microplus fed on a calf infected with the strain Jaboticabal of A. marginale. At the end of the parasitic phase, fully engorged females were incubated at either 18°C or 28°C for oviposition. Although A. marginale was detected in the salivary glands of the females, demonstrating that the ticks were successfully infected, the presence of rickettsia was not detected in the offspring. However, the preoviposition period of the non-infected females maintained at 18°C was longer than that of those maintained at 28°C. In addition, the average weight of the mass of eggs as well as the egg production efficiency (ratio of the egg mass weight to the female weight) of the females maintained at 18°C were significantly lower than those of the females incubated at 28°C. There was no larval hatching from the eggs maintained exclusively at 18°C, even at 65 days after female detachment. Hatching occurred only when the eggs maintained at 18°C were transferred to 28°C at 20 days after female detachment (18°C/28°C). We also verified a significantly higher larvae conversion efficiency (ratio of the larvae mass weight to the egg mass weight) in the group of females maintained exclusively at 28°C compared to those from the 18°C/28°C group. Collectively, our results reinforce that low temperature exerts negative effects on female fertility and egg development in R. microplus, although it has no influence on A. marginale transmission to the progeny. In the field, the detrimental effects of temperatures on tick reproductive fitness lead to a reduction of tick population, which may cause a decrease in the incidence of bovine anaplasmosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The taxonomic composition of egg-associated microbial communities can play a crucial role in the development of fish embryos. In response, hosts increasingly influence the composition of their associated microbial communities during embryogenesis, as concluded from recent field studies and laboratory experiments. However, little is known about the taxonomic composition and the diversity of egg-associated microbial communities within ecosystems; e.g., river networks. We sampled late embryonic stages of naturally spawned brown trout at nine locations within two different river networks and applied 16S rRNA pyrosequencing to describe their bacterial communities. We found no evidence for a significant isolation-by-distance effect on the composition of bacterial communities, and no association between neutral genetic divergence of fish host (based on 11 microsatellites) and phylogenetic distances of the composition of their associated bacterial communities. We characterized core bacterial communities on brown trout eggs and compared them to corresponding water samples with regard to bacterial composition and its presumptive function. Bacterial diversity was positively correlated with water temperature at the spawning locations. We discuss this finding in the context of the increased water temperatures that have been recorded during the last 25 years in the study area.

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb uses the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT), which induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential coenzyme NAD(+). Expression or injection of a noncatalytic TNT mutant showed no cytotoxicity in macrophages or in zebrafish zygotes, respectively, thus demonstrating that the NAD(+) glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT-IFT complex revealed a new NAD(+) glycohydrolase fold of TNT, the founding member of a toxin family widespread in pathogenic microorganisms.

Background: The mechanical transmission of pathogenic bacteria by synanthropic filth flies is widely recognized. While many studies report the fate and the temporospatial distribution of ingested foodborne bacteria by filth flies, there is little evidence about the transmission dynamics of ingested foodborne bacteria by adult house flies (Musca domestica) to their progeny. In this study, we fed parental house fly adults with food contaminated with low, medium, and high concentrations of Salmonella enterica, Cronobacter sakazakii, Escherichia coli O157:H7, and Listeria monocytogenes and evaluated the probability of transmission of these pathogens to house fly eggs and the surface and the alimentary canal of their first filial (F1) generation adults., Results: All foodborne pathogens were present in samples containing pooled house fly eggs. The probability of transmission was higher after parental house flies ingested food containing medium bacterial loads. Cronobacter sakazakii was 16, 6, and 3 times more likely to be transmitted to house fly eggs than S. enterica, E. coli O157:H7, and L. monocytogenes, respectively. Only S. enterica and C. sakazakii were transmitted to F1 generation adults and their presence was 2.4 times more likely on their body surfaces than in their alimentary canals. The highest probabilities of finding S. enterica (60 %) and C. sakazakii (28 %) on newly emerged F1 adults were observed after parental house flies ingested food containing medium and high levels of these pathogens, respectively., Conclusion: Our study demonstrates that adult house flies that fed from food contaminated with various levels of foodborne bacteria were able to transmit those pathogens to their eggs and some were further transmitted to newly emerged F1 generation adults, enhancing the vector potential of these insects. Understanding the type of associations that synanthropic filth flies establish with foodborne pathogens will help to elucidate transmission mechanisms and possible ways to mitigate the spread of foodborne pathogens.

Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A facultative anaerobe, alkalitolerant, gram-negative marine bacterium strain LBS5(T), was isolated from eggs carried on the pleopods of female spiny lobster (Panulirus penicillatus) in Andaman Sea from a depth of 3.5 m. Heterotrophic growth was observed at 15-38 °C and pH 5.5-11. Optimum growth occurred at 28 °C and pH 7.5. It can grow in the presence of 0.5-7 % NaCl (w/v), and the optimal NaCl required for growth was 2-4 %. 16S rRNA gene sequence analysis revealed the strain LBS5(T) belongs to the genus Photobacterium and showed 99.6 % similarity with P. aquae AE6(T), 98.2 % with P. aphoticum M46(T), 97 % with P. rosenbergii CC1(T), 96.9 % with P. lutimaris DF-42(T), and 96.6 % with P. halotolerans MACL01(T). The DNA-DNA similarities between strains LBS5(T) with other closely related strains were well below 70 %. The DNA G + C content was 50.52 (±0.9) mol%. The major fatty acids were C16:1w7c/w6c, C18:1w6c/w7c, C16:0, C15:0 iso, C16:0 10-methyl/17:1 iso w9c, C17:0 iso. Polar lipids included a phosphatidylglycerol, a diphosphatidylglycerol, a phosphatidylethanolamine, and one unidentified lipid. Based on the polyphasic evidences, strain LBS5(T) represents a novel species of the genus Photobacterium for which Photobacterium panuliri sp. nov. is proposed. The type strain is LBS5(T) (=DSM 27646(T) = LMG 27617(T) = JCM 19199(T)).

Research involving the use of nematophagous fungi in the biological control of parasites of interest to veterinarians has occurred over recent years, with promising results. This article reports the infection of Parascaris equorum eggs by the fungus Pochonia chlamydosporia (isolates VC1 and VC4). Six groups were formed for each isolate, with six different culture media: 2% water-agar (2% WA); agar-chitin (AC); YPSSA (yeast extract, K2HPO4, MgSO4 ·7H2O, soluble starch); AELA extract (starch + water + agar); 2% corn-meal-agar (2% CMA); and 2% potato dextrose-agar (2% PDA). A total of 1000 eggs of P. equorum were transferred to each plate containing isolates grown for a period of 7 days (treatment group). Also, 1000 eggs were added to each plate without fungus (controlgroup). The plates were kept in an environmental chamber at 25 °C in the dark for 21 days. After, we analyzed the effects on ovicidal activity: effect 1 (accession shell); effect 2 (penetration hyphae); and effect 3 (destruction of the eggs). No differences were observed in the destruction of eggs between the two isolates. The decreasing effectiveness of the different culture media was: PDA (38.9%); CMA (38.3%); WA (36.7%); YPSSA (36.45%); and AC (32.5%). The highest percentage egg destruction was observed when the strains were grown in culture medium AELA (44.9%); this was the best medium., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ovicidal activity of seven fungal strains: Acremonium alabamense, Alternaria chlamydospora, Cladosporium herbarum, Fusarium solani, Paecilomyces variotii, Paecilomyces viridis and Penicillium verruculosum isolated from urban soil samples from Poland was determined in vitro. The fungal mycelium was co-cultured with Ascaris suum eggs on plates with 2% water-agar for 28 days. Eggs exposed and unexposed (control) to fungal mycelium were observed weekly by light microscopy and the percentage of malformed eggs were determined. The eggs were classified according to following parameters: type 1 - biochemical and physiological effect without morphological damage to the eggshell; type 2 - lytic effect with morphological alteration of the eggshell and embryo; type 3 - lytic effect with morphological alteration of eggshell and embryo with hyphal penetration and internal egg colonization. All examined species of fungi extended embryogenesis, but the retardation of embryonic development was varied and depended on the species. A. alabamense, A. chlamydospora and P. verruculosum exhibited very high inhibitory activity on A. suum egg development. The fungus-exposed eggs revealed morphological alternations in all stages of embryogenesis. Isolates of F. solani, P. variotii and P. viridis showed hyphal penetration and internal colonization of A. suum eggs (type 3 effect). No appressoria were produced and simple hyphal penetrations were most commonly observed. A. alabamense and P. verruculosum demonstrated morphological destruction, with eggshell destruction. The remaining fungi showed type 1 effect. The results demonstrated that examined strains of F. solani, P. variotii and P. viridis may be considered to be potential limiting factors of parasitic geohelminth populations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The resident gut flora is known to have significant impacts on the life history of the host organism. Endosymbiotic bacterial species in the Anopheles mosquito gut are potent modulators of sexual development of the malaria parasite, Plasmodium, and thus proposed as potential control agents of malaria transmission., Results: Here we report a melanotic pathology in the major African malaria vector Anopheles gambiae, caused by the dominant mosquito endosymbiont Elizabethkingiameningoseptica. Transfer of melanised tissues into the haemolymph of healthy adult mosquitoes or direct haemolymph inoculation with isolated E. meningoseptica bacteria were the only means for transmission and de novo formation of melanotic lesions, specifically in the fat body tissues of recipient individuals. We show that E. meningoseptica can be vertically transmitted from eggs to larvae and that E. meningoseptica-mono-associated mosquitoes display significant mortality, which is further enhanced upon Plasmodium infection, suggesting a synergistic impact of E. meningoseptica and Plasmodium on mosquito survival., Conclusion: The high pathogenicity and permanent association of E. meningoseptica with An. Gambiae through vertical transmission constitute attractive characteristics towards the potential design of novel mosquito/malaria biocontrol strategies.

Bacteria of the genus Aeromonas are found worldwide in aquatic environments and may produce human infections. In 2010, two new clinical species, Aeromonas sanarellii and Aeromonas taiwanensis, were described on the basis of one strain recovered from wounds of hospitalized patients in Taiwan. So far, only four environmental isolates of A. sanarellii and one of A. taiwanensis have been recorded from waste water in Portugal and an additional clinical strain of A. taiwanensis from the faeces of a patient with diarrhoea in Israel. In the present study, strains belonging to these two species were identified from chironomid egg masses from the same area in Israel by sequencing the rpoD gene. This represents a new environmental habitat for these novel species. The first data on the virulence genes and antibiotic susceptibility are provided. The isolates of these two new species possess multiple virulence genes and are sensitive to amikacin, aztreonam, cefepime, cefoxatime, ceftazidime, ciprofloxacin, gentamicin, piperacillin-tazobactam, tigecycline, tobramycin, trimethoprim-sulfamethoxazole and imipenem. The key phenotypic tests for the differentiation of these new species from their closest relative Aeromonas caviae included the utilization of citrate, growth at 45 °C in sheep blood agar and acid production of cellobiose., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances.

Eukaryotic organisms carry various genetic factors the so-called cytoplasmic genetic elements (CGEs), in their cytoplasm. Numerous examples are known in which CGEs possess the ability to control sex determination of their host organisms and cause sex ratio distortion (SRD). In general, CGEs are inherited maternally from female hosts, via egg cytoplasm to offspring. Thus, the elements tend to evolve abilities to avoid entrance into "dead-end" males. Previous theoretical studies have revealed that, as long as maternal transmission is perfect, CGEs evolve the highest levels of ability to cause SRD. However, it is recently reported that some CGEs transmit from male to offspring through infection to female in mating. This raises the question of how such a paternal contribution alters selective forces and SRD evolution. In the present study, the evolutionary process of SRD ability of CGEs was analyzed theoretically. The main finding is that paternal transmission results in evolution towards intermediate levels of SRD. Further, coexistence was observed of different CGEs inducing different levels of SRD. These results point to the importance of paternal transmission in the evolution of CGEs., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The effect of two disinfectants on eggs and larvae of Baltic cod, Gadus morhua, was investigated. The eggs were disinfected for 10 min using various concentrations of either glutaraldehyde (100, 200, 400, 600 and 800 mg L(-1)) or iodophor (10, 50, 100 and 150 mg L(-1)), 1-4-days post-fertilization. Bactericidal effect of disinfection, survival to hatching, hatching success and larval abnormalities were assessed. Larval survival was recorded at 5-, 10- and 15-days post-hatch (dph). Although Baltic cod eggs have an unusually thin chorion, they could tolerate surface disinfection. A reduction in bacterial growth was observed with increased concentrations of disinfectant (3.0 x 10(7)-1.6 x 10(1) CFU mL(-1)). Abnormalities in newly hatched larvae were not related to disinfection. Survival of the yolk sac larvae was significantly better for eggs treated with 400 mg L(-1) glutaraldehyde for 10 min at 10 and 15 dph. Effective disinfection was also recorded using 100 mg L(-1) Actomar K30. Egg batch effect rather than initial bacterial concentration, disinfectant type or incubation method determined the survival of the eggs to hatching and survival of larvae. Because of the carcinogenic effect of glutaraldehyde, iodophor is recommended for routine disinfection of cod eggs.

An assessment was made of the ovicidal activity of egg-parasitizing fungi Pochonia chlamydosporia (isolates VC1 and VC4) and Paecilomyces lilacinus on Toxocara canis eggs in vitro. The fungal isolates were inoculated onto Petri dishes with 2% water-agar (2% WA) and stored at 25 degrees C for 10 days in an incubator, in the dark. The control group was comprised of Petri dishes without fungi, containing the 2%WA medium only. Later, 1000 embryonated eggs were placed on the surface of the plates with fungal isolates and also on the control plates, and were then incubated at 25 degrees C for 7, 14 and 21 days. At these intervals, the eggs were retrieved and underwent percentage assessment according to the following parameters: no changes; type 1 effect, physiological and biochemical effect without morphological damage to eggshell, with visualization of hyphae adhered to eggshell; type 2 effect, lytic effect with morphological changes in embryo and eggshell, without hyphal penetration through the eggshell; type 3 effect, lytic effect with morphological changes in embryo and eggshell, with hyphal penetration and internal egg colonization. All the fungal isolates showed ovicidal activity (type 3 effect) on T. canis eggs, with 13.8%, 20.5% and 20.3% of ovicidal activity using P. chlamydosporia isolate VC1 after 7, 14 and 21 days, whereas isolate VC4 showed 15.2%, 19.0% and 21.7% of ovicidal activity at the same time intervals. P. lilacinus showed ovicidal activity of 12.3%, 18.8% and 20.0% after 7, 14 and 21 days. P. chlamydosporia and P. lilacinus were effective in vitro on T. canis eggs and can be considered a potential candidate to biological controller of those nematodes.

Sea turtles migrate to various habitats where they can be exposed to different pollutants. Bacteria were collected from turtle eggs and their resistance to antibiotics was used as pollutant bio-indicators of contaminated effluents. Eggs were collected randomly from turtles when they were laying their eggs. A total of 90 eggs were collected and placed into sterile plastic bags (3 eggs/turtle) during June-December of 2003. The bacteria located in the eggshell, albumen and yolk were examined, and 42% of the eggs were contaminated with 10 genera of bacteria. Pseudomonas spp. were the most frequent isolates. The albumen was found to be the part of the egg to be the least contaminated by bacterial infection. Bacterial isolates tested with 14 antibiotics showed variations in resistance. Resistance to ampicillin was the highest. The presence of antibiotic resistant bacteria in eggs indicates that the green turtle populations were subjected to polluted effluents during some of their migratory routes and feeding habitats. Scanning electron microscopy revealed that Salmonella typhimurium penetrated all eggshell layers.

Purpose: The present study focused on the effect of two fungal species Metarhizium flavoviride and Metarhizium anisopliae on the embryonic development and viability of Ascaris suum larvae., Results: Microscope examination revealed that the presence of the mycelium inhibited the egg development, compared to control. The fungus-exposed eggs featured zygote vacuolisation, irregular blastomere cleavage, and morphological disturbances in the stages of blastula, gastrula, and larva. The embryopathies were significantly more frequent in the eggs incubated with M. flavoviride. Also the mortality of the developed larvae was significantly higher after 60-day culture with M. flavoviride.

Although Pseudomonas fluorescens was the predominant bacterium associated with Atlantic salmon (Salmo salar) eggs incubated at the White River National Fish Hatchery (Bethel, Vermont) during January 1992, the fish pathogen Cytophaga psychrophila was isolated only from specific lots of eggs that displayed poor survival (35% eye-up).

Objective: Our long-term objective is to determine the consequences of HIV-1 expression in the mammalian fetus using the mouse as a model system. This study describes HIV gene expression in micro-injected and electroporated embryos during the pre-implantation period., Design: Procedures were adopted to create a non-infectious HIV system to assay HIV long terminal repeat (LTR) activation in a non-human mammal., Methods: Two constructs (RSV promoter-HIV tat gene; HIV LTR-lac Z gene) were co-micro-injected (10 micrograms/ml each) into pronuclei of fertilized eggs or one nucleus of the two-cell stage and expression assessed after 2-5 days. The techniques of electroporation and micro-injection were compared for their ability to deliver the constructs expressed subsequently., Results: We found that HIV-1 expression, as monitored by beta-galactosidase production, can occur as early as the eight-cell to blastocyst stage. The intensity of HIV expression varied from high to low; the proportion of the embryo expressing HIV varied from half to only two cells. The material was too limited to allow a determination of integration., Conclusions: The expression rate of the HIV-LTR was low, averaging slightly over 1%. The data demonstrate that the HIV-LTR can be activated in early mammalian development, even before implantation. This information is valuable to those studying HIV-transgenic mice, to those interested in factors governing HIV expression, and to those concerned about pathologies accompanying developmental expression of HIV.

Following a definition of bacterial L-forms and a literature review on introduction and reversion of bacteria to and from L-forms, respectively, the results of a study comprising 587 samples (316 pigeons and 271 pigeon eggs) are reported. As a control 25 free living pigeons and 25 eggs of those pigeons were used, because antibiotic treatment of these could be excluded. From 79 samples (75 pigeons and 4 eggs) Salmonella typhimurium var. copenhagen (STMC) was isolated (none from the urban pigeons), of them 11 in the L-form from joints and organs and 3 from eggs. 325 of other bacterial isolates were found as L-forms (= approx. 40%). Out of 168 serum samples investigated, 33.9% showed antibodies against STMC. Corresponding antibodies could only be demonstrated in 73.4% of the pigeons with a STMC isolate. The occurrence of L-forms explains resistance to therapy and the failure of vaccines. The high frequency of L-forms is probably an indication of an inconsequential use of antibiotics in managing pigeon diseases.

Mouse two-cell embryos, morulae, and blastocysts were killed when infected in vitro with simian virus 40 (SV40) at high multiplicities of infection. Polyoma virus was not deleterious for preimplantation embryos, even at a very high multiplicity of infection; however, the outgrowths of polyoma-infected blastocysts disintegrated after several days of culture. Indirect immunofluorescence tests revealed the presence of SV40 T and V antigens and polyoma virus V antigen in the nuclei of trophoblastic cells. Virus-specific antigens were not found in the nuclei of cells forming inner cell masses of blastocysts or in inner cell mass-derived cells in blastocyst out-growths. The appearance of SV40 T and V antigens in the nuclei was inhibited by alphaamanitin, a RNA polymerase II inhibitor. The amount of infectious virus recovered from cultures of morulae or blastocysts on subsequent days after infection with SV40 initially declined but later increased. These points of evidence indicate that some cells of early mouse embryos are permissive for the expression of early and late functions of SV40 genome and that susceptibility to infection with polyoma virus and/or permissiveness for the expression of polyoma virus late functions develop gradually between the two-cell and blastocyst stages. Electron microscope observations showed the presence of specific complexes of membranes and virions in the cytoplasm of trophoblastic cells. Single viral particles could be found in the nuclei and also in mitochondria.

Chlamydia trachomatis (strains L2 and DE) were found to infect fertilized mouse ova in vitro. Inclusions were found in the trophoblast, but not in the embryo.

Rous associated virus type-0 (RAV-0), a subgroup E replication-competent endogenous virus of chickens, is associated with a low efficiency of virus shedding into the egg albumen and failure to establish congenital transmission. In contrast, RAV-1, a subgroup A virus of exogenous origin, is efficiently shed into the albumen and readily infects the embryo. Among a series of in vitro constructed recombinants between RAV-0 and RAV-1, we have identified subgroup E recombinants that efficiently shed virus into the egg albumen but do not undergo efficient congenital transmission. The LTR region, subgroup-determining sequences in env, and sequences within a 375 bp Sacl-Xhol fragment at the 5' end of the genome each influenced the efficiency of virus shedding into the albumen. Egg inoculations with viruses differing only in env were used to confirm the low rate of congenital transmission of subgroup E viruses. These studies revealed that subgroup A envelope antigens are at least 100-fold more effective for the establishment of embryonic infection than subgroup E.

The paper covers experimental results of introducing exogenic genetic material, namely DNA sequences of the Rous sarcoma virus, by microinjections in mice zygotes and Drosophila early embryos. In a number of cases integration of viral DNA into genomes of these organisms was detected. Blot-hybridizations analysis of cell DNA proved that the inserted viral sequences undergo rearrangements in the course of integration.