A taste of a toxin paradise: Xenorhabdus and Photorhabdus bacterial secondary metabolites against Aedes aegypti larvae and eggs.

Aedes-transmitted arboviral infections such as Dengue, Yellow Fever, Zika and Chikungunya are increasing public health problems. Xenorhabdus and Photorhabdus bacteria are promising sources of effective compounds with important biological activities. This study investigated the effects of cell-free supernatants of X. szentirmaii, X. cabanillasii and P. kayaii against Ae. aegypti eggs and larvae and identified the bioactive larvicidal compound in X. szentirmaii using The EasyPACId method. Among the three tested bacterial species, X. cabanillasii exhibited the highest (96%) egg hatching inhibition and larvicidal activity (100% mortality), whereas P. kayaii was the least effective species in our study. EasyPACId method revealed that bioactive larvicidal compound in the bacterial supernatant was fabclavine. Fabclavines obtained from promoter exchange mutants of different bacterial species such as X. cabanillasii, X. budapestensis, X. indica, X. szentirmaii, X. hominckii and X. stockiae were effective against mosquito larvae. Results show that these bacterial metabolites have potential to be used in integrated pest management (IPM) programmes of mosquitoes.

Study on the virulence of Metarhizium anisopliae against Spodoptera frugiperda (J. E. Smith, 1797).

This study examined the impact of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) conidia on the eggs, larvae, pupae, and adults of Spodoptera frugiperda. The results showed that eggs, larvae, pupae, and adults exhibited mortality rates that were dependent on the dose. An increased amount of conidia (1.5 × 109 conidia/mL) was found to be toxic to larvae, pupae, and adults after 9 days of treatment, resulting in a 100% mortality rate in eggs, 98% in larvae, 76% in pupae, and 85% in adults. A study using earthworms as bioindicators found that after 3 days of exposure, M. anisopliae conidia did not cause any harmful effects on the earthworms. In contrast, the chemical treatment (positive control) resulted in 100% mortality at a concentration of 40 ppm. Histopathological studies showed that earthworm gut tissues treated with fungal conidia did not show significant differences compared with those of the negative control. The gut tissues of earthworms treated with monocrotophos exhibited significant damage, and notable differences were observed in the chemical treatment. The treatments with 70 and 100 µg/mL solutions of Eudrilus eugeniae epidermal mucus showed no fungal growth. An analysis of the enzymes at a biochemical level revealed a decrease in the levels of acetylcholinesterase, α-carboxylesterase, and ß-carboxylesterase in S. frugiperda larvae after exposure to fungal conidia. This study found that M. anisopliae is effective against S. frugiperda, highlighting the potential of this entomopathogenic fungus in controlling this agricultural insect pest.

First evidence of the activity of an entomopathogenic fungus against the eggs of Sarcoptes scabiei.

The entomopathogenic fungus Beauveria bassiana has been successfully used for the control of phytopathogenic arthropods and there are a growing number of studies suggesting that this kind of fungus could also be used for the control of ectoparasites in mammals. This study evaluated for the first time the efficacy of different Beauveria strains against the eggs of Sarcoptes scabiei collected from experimentally infected pigs. Eggs were exposed to fungal conidia and monitored for hatching over 10 days. The strongest effect (28.75 % of hatching inhibition) was obtained with a commercial B. bassiana strain. Furthermore, the detection of fungal genomic within the surface-cleaned eggs demonstrated the ability of B. bassiana to penetrate and proliferate in the egg-shell of S. scabiei. This study provides the first evidence, using molecular techniques, that the development of mycoacaricides may be of interest for the control of S. scabiei infection.

Development of Metarhizium humberi in Aedes aegypti eggs.

The entomopathogenic fungus Metarhizium humberi affects Aedes aegypti adults, larvae and eggs, but its ovicidal activity is not yet well documented. Conidia of this fungus adhered to the chorion, initiated germination within 12 h, and germinating conidia were detected for up to 10 d after contact with the egg. Germ tubes either penetrated the chorion directly or formed appressoria at the end of a short hypha (<5 µm) or, subsequently, on longer, branched hyphae. Thin layers of what was most probably a fungal mucilaginous excretion were detected on the chorion adjacent to germ tubes, appressoria and hyphae. After 5 d eggs frequently appeared shriveled with ruptures in the chorion, and with the interior filled with hyphae that eventually produced mycelium and new conidia on the egg surfaces. Findings demonstrated that this fungus can infect A. aegypti eggs and subsequently recycle on their surface by producing large numbers of new conidia that should be infective for further generations of eggs, larvae and adults.

Ovicidal Efficacy of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) towards Rhipicephalus sanguineus (Acari: Ixodidae) Eggs.

Rhipicephalus sanguineus, commonly known as brown dog tick is a widespread species with considerable public health and economic importance. Tremendous efforts were performed to control the tick populations with the concern of resistance build-up and environmental issues. Alternative towards microbial control thus emerged as one option to reduce tick populations. In this study, the ovicidal efficacy of a native isolate entomopathogenic hyphomycetes fungi, Metarhizium anisopliae strain HSAH5 was evaluated against eggs of R. sanguineus. Spray applications with three different conidial concentrations of 105, 106 and 107 conidia mL-1; 40 ppm of Flumethrin and a negative control. The M. anisopliae strain was found highly virulent to R. sanguineus eggs by reducing the hatching percentages to ≈30% compared with 8.9% in Flumethrin eggs. The result showed a significantly higher mortality in M. anisopliae group than those of the control groups (F = 42.08, df = 32, P < 0.001) at 30 days post-infection. However, there are no significant differences within the M. anisopliae group, in which the mortality between different conidial concentrations is almost the same. The estimated LC50 of M. anisopliae against eggs of R. sanguineus is 1.36 × 103 conidia ml-1. Thus, these results suggest M. anisopliae strain HSAH5 could be a potential biocontrol agent of R. sanguineus in the integrated approach to managing ticks in the residential landscape by targeting on the eggs.

Symbiont Genomic Features and Localization in the Bean Beetle Callosobruchus maculatus.

A pervasive pest of stored leguminous products, the bean beetle Callosobruchus maculatus (Coleoptera: Chrysomelidae) associates with a simple bacterial community during adulthood. Despite its economic importance, little is known about the compositional stability, heritability, localization, and metabolic potential of the bacterial symbionts of C. maculatus. In this study, we applied community profiling using 16S rRNA gene sequencing to reveal a highly conserved bacterial assembly shared between larvae and adults. Dominated by Firmicutes and Proteobacteria, this community is localized extracellularly along the epithelial lining of the bean beetle's digestive tract. Our analysis revealed that only one species, Staphylococcus gallinarum (phylum Firmicutes), is shared across all developmental stages. Isolation and whole-genome sequencing of S. gallinarum from the beetle gut yielded a circular chromosome (2.8 Mb) and one plasmid (45 kb). The strain encodes complete biosynthetic pathways for the production of B vitamins and amino acids, including tyrosine, which is increasingly recognized as an important symbiont-supplemented precursor for cuticle biosynthesis in beetles. A carbohydrate-active enzyme search revealed that the genome codes for a number of digestive enzymes, reflecting the nutritional ecology of C. maculatus. The ontogenic conservation of the gut microbiota in the bean beetle, featuring a "core" community composed of S. gallinarum, may be indicative of an adaptive role for the host. In clarifying symbiont localization and metabolic potential, we further our understanding and study of a costly pest of stored products. IMPORTANCE From supplementing essential nutrients to detoxifying plant secondary metabolites and insecticides, bacterial symbionts are a key source of adaptations for herbivorous insect pests. Despite the pervasiveness and geographical range of the bean beetle Callosobruchus maculatus, the role of microbial symbioses in its natural history remains understudied. Here, we demonstrate that the bean beetle harbors a simple gut bacterial community that is stable throughout development. This community localizes along the insect's digestive tract and is largely dominated by Staphylococcus gallinarum. In elucidating symbiont metabolic potential, we highlight its possible adaptive significance for a widespread agricultural pest.

Development of a wild-type Escherichia coli environmental bloom model to evaluate alternatives to formaldehyde fumigation in broiler chicken hatch cabinets.

Horizontal transmission of opportunistic Escherichia coli during hatch can have detrimental effects on early performance, particularly as pioneer colonizers. Commercially, formaldehyde is often applied in the United States to combat the bacterial bloom that occurs inside of the hatching environment. The purpose of these experiments was to develop a replicable E. coli horizontal challenge model to evaluate alternatives to formaldehyde sanitation applied to the hatching environment. In experiment 1, two trials were conducted for 2 wild-type (WT) E. coli isolates (isolate 1 [I1] or isolate 2 [I2]) to determine the appropriate in ovo challenge dose and day of embryogenesis (DOE) for challenge administration. In experiment 1 trial 1, the most appropriate inoculation dose and time point were determined to be 102 cfu/embryo on DOE 19. Experiment 1 trial 2 evaluated whether placement of seeder (direct-challenged) embryos with contact (indirect-challenged) embryos during hatch affected contact hatchability. Trial 2 showed no differences in hatchability between groups. A 7-day experiment (experiment 1 trial 2) was conducted to evaluate the effects of I1 or I2 on horizontal transmission, gram-negative bacterial (GNB) recovery from the gastrointestinal tract (GIT), and impact on BW gain (BWG). Compared with the negative control, seeder, and contact chicks challenged with I1 or I2, we observed increased (P < 0.05) GNB recovered from GIT on the day of hatch. There was a marked (P < 0.05) reduction in 7-day BWG between the I1 indirect-challenged group and the negative control group. To further validate the model, 2 7-day trials (experiment 2, experiment 3) were conducted to evaluate the effects of formaldehyde fumigation on coliform recovery from the hatching environment and on early performance using I1 for the challenge. Isolate 1 positive control hatchers had increased levels of circulating coliforms compared with the negative control and formaldehyde-treated hatchers, although there was no significant impact on performance induced by challenge or formaldehyde treatment in experiment 2 or experiment 3. These data provide a potential model for investigations related to horizontal transmission of WT E. coli at a low dose on DOE 19 to promote simulated commercially relevant bacterial blooms under laboratory conditions.

Disentangling the Relative Roles of Vertical Transmission, Subsequent Colonizations, and Diet on Cockroach Microbiome Assembly.

A multitude of factors affect the assemblies of complex microbial communities associated with animal hosts, with implications for community flexibility, resilience, and long-term stability; however, their relative effects have rarely been deduced. Here, we use a tractable lab model to quantify the relative and combined effects of parental transmission (egg case microbiome present/reduced), gut inocula (cockroach versus termite gut provisioned), and varying diets (matched or unmatched with gut inoculum source) on gut microbiota structure of hatchlings of the omnivorous cockroach Shelfordella lateralis using 16S rRNA gene (rDNA) amplicon sequencing. We show that the presence of a preexisting bacterial community via vertical transmission of microbes on egg cases reduces subsequent microbial invasion, suggesting priority effects that allow initial colonizers to take a strong hold and which stabilize the microbiome. However, subsequent inoculation sources more strongly affect ultimate community composition and their ecological networks, with distinct host-taxon-of-origin effects on which bacteria establish. While this is so, communities respond flexibly to specific diets in ways that consequently impact predicted community functions. In conclusion, our findings suggest that inoculations drive communities toward different stable states depending on colonization and extinction events, through ecological host-microbe relations and interactions with other gut bacteria, while diet in parallel shapes the functional capabilities of these microbiomes. These effects may lead to consistent microbial communities that maximize the extended phenotype that the microbiota provides the host, particularly if microbes spend most of their lives in host-associated environments.IMPORTANCE When host fitness is dependent on gut microbiota, microbial community flexibility and reproducibility enhance host fitness by allowing fine-tuned environmental tracking and sufficient stability for host traits to evolve. Our findings lend support to the importance of vertically transmitted early-life microbiota as stabilizers, through interactions with potential colonizers, which may contribute to ensuring that the microbiota aligns within host fitness-enhancing parameters. Subsequent colonizations are driven by microbial composition of the sources available, and we confirm that host-taxon-of-origin affects stable subsequent communities, while communities at the same time retain sufficient flexibility to shift in response to available diets. Microbiome structure is thus the result of the relative impact and combined effects of inocula and fluctuations driven by environment-specific microbial sources and digestive needs. These affect short-term community structure on an ecological time scale but could ultimately shape host species specificities in microbiomes across evolutionary time, if environmental conditions prevail.

Diversity of bacteria in different life stages and their impact on the development and reproduction of Zeugodacus tau (Diptera: Tephritidae).

Fruit flies usually harbor diverse communities of bacteria in their digestive systems, which are known to play a significant role in their fitness. However, little information is available on Zeugodacus tau, a polyphagous pest worldwide. This study reports the first extensive analysis of bacterial communities in different life stages and their effect on the development and reproduction of laboratory-reared Z. tau. Cultured bacteria were identified using the conventional method, and all bacteria were identified by high-throughput technologies (16S ribosomal RNA gene sequencing of V3-V4 region). A total of six bacterial phyla were identified in larvae, pupae, and male and female adult flies, which were distributed into 14 classes, 32 orders, 58 families and 96 genera. Proteobacteria was the most represented phylum in all the stages except larvae. Enterobacter, Klebsiella, Providencia, and Pseudomonas were identified by conventional and next-generation sequencing analysis in both male and female adult flies, and Enterobacter was found to be the main genus. After being fed with antibiotics from the first instar larvae, bacterial diversity changed markedly in the adult stage. Untreated flies laid eggs and needed 20 days before oviposition while the treated flies showed ovary development inhibited and were not able to lay eggs, probably due to the alteration of the microbiota. These findings provide the cornerstone for unexplored research on bacterial function in Z. tau, which will help to develop an environmentally friendly management technique for this kind of harmful insect.

Microbial Communities of Stored Product Mites: Variation by Species and Population.

Arthropod-associated microorganisms are important because they affect host fitness, protect hosts from pathogens, and influence the host's ability to vector pathogens. Stored product mites (Astigmata) often establish large populations in various types of food items, damaging the food by direct feeding and introducing contaminants, including their own bodies, allergen-containing feces, and associated microorganisms. Here we access the microbial structure and abundance in rearing diets, eggs, feces fraction, and mite bodies of 16 mite populations belonging to three species (Carpoglyphus lactis, Acarus siro, and Tyrophagus putrescentiae) using quantitative PCR and 16S ribosomal RNA (rRNA) gene amplicon sequencing. The mite microbiomes had a complex structure dominated by the following bacterial taxa (OTUs): (a) intracellular symbionts of the genera Cardinium and Wolbachia in the mite bodies and eggs; (b) putative gut symbionts of the genera Solitalea, Bartonella, and Sodalis abundant in mite bodies and also present in mite feces; (c) feces-associated or environmental bacteria of the genera Bacillus, Staphylococcus, and Kocuria in the diet, mite bodies, and feces. Interestingly and counterintuitively, the differences between microbial communities in various conspecific mite populations were higher than those between different mite species. To explain some of these differences, we hypothesize that the intracellular bacterial symbionts can affect microbiome composition in mite bodies, causing differences between microbial profiles. Microbial profiles differed between various sample types, such as mite eggs, bodies, and the environment (spent growth medium-SPGM). Low bacterial abundances in eggs may result in stochastic effects in parent-offspring microbial transmission, except for the intracellular symbionts. Bacteria in the rearing diet had little effect on the microbial community structure in SPGM and mite bodies. Mite fitness was positively correlated with bacterial abundance in SPGM and negatively correlated with bacterial abundances in mite bodies. Our study demonstrates critical host-microbe interactions, affecting all stages of mite growth and leading to alteration of the environmental microbiome. Correlational evidence based on absolute quantitation of bacterial 16S rRNA gene copies suggests that mite-associated microorganisms are critical for modulating important pest properties of mites by altering population growth.

ß-Cyclodextrin Encapsulation of Synthetic AHLs: Drug Delivery Implications and Quorum-Quenching Exploits.

Many bacteria, such as Pseudomonas aeruginosa, regulate phenotypic switching in a population density-dependent manner through a phenomenon known as quorum sensing (QS). For Gram-negative bacteria, QS relies on the synthesis, transmission, and perception of low-molecular-weight signal molecules that are predominantly N-acyl-l-homoserine lactones (AHLs). Efforts to disrupt AHL-mediated QS have largely focused on the development of synthetic AHL analogues (SAHLAs) that are structurally similar to native AHLs. However, like AHLs, these molecules tend to be hydrophobic and are poorly soluble under aqueous conditions. Water-soluble macrocycles, such as cyclodextrins (CDs), that encapsulate hydrophobic guests have long been used by both the agricultural and pharmaceutical industries to overcome the solubility issues associated with hydrophobic compounds of interest. Conveniently, CDs have also demonstrated anti-AHL-mediated QS effects. Here, using fluorescence spectroscopy, NMR spectrometry, and mass spectrometry, we evaluate the affinity of SAHLAs, as well as their hydrolysis products, for ß-CD inclusion. We also evaluated the ability of these complexes to inhibit wild-type P. aeruginosa virulence in a Caenorhabditis elegans host infection study, for the first time. Our efforts confirm the potential of ß-CDs for the improved delivery of SAHLAs at the host/microbial interface, expanding the utility of this approach as a strategy for probing and controlling QS.

The use of embryonic chicken eggs as an alternative model to evaluate the virulence of Salmonella enterica serovar Gallinarum.

Salmonella enterica serovar Gallinarum (S. Gallinarum) can cause fowl typhoid, a severe systemic disease responsible for considerable economic losses. Chicken pathogenicity test is the traditional method for assessing the virulence of S. Gallinarum. However, this method is limited by several factors, including ethical considerations, costs, and the need for specialized facilities. Hence, we established a chicken embryo lethality assay (ELA) model to determine the virulence of S. Gallinarum. Three virulent and three avirulent representative strains, which were confirmed by the chicken pathogenicity test, were used to perform the ELA. The most significant difference between the virulent and avirulent strains could be observed when 13-day-old embryos were inoculated via the AC route and incubated for 5 days. Based on a 50% embryo lethal dose (ELD50), isolates considered to be virulent had a Log10ELD50 of ≤ 4.0, moderately virulent strains had a Log10ELD50 of 4.0-6.1, and avirulent isolates had a Log10ELD50 of ≥ 6.1. Different abilities to invade the liver of embryos were found between the virulent and avirulent strains by a growth curve experiment in vitro. The maximum colony-forming units (CFU) of the virulent strain was about 10,000 times higher than that of the avirulent strain in the liver at 5 days post infection. The ELA results of 42 field strains showed that thirty-two strains (76.2%) were virulent, nine were moderately virulent (21.4%), and one strain was avirulent (2.4%). In conclusion, these results suggest that the ELA can be used as an alternative method to assess the virulence of S. Gallinarum, which will contribute to the study of virulence genes, virulence evolution, pathogenic mechanisms and vaccine development.

Egg-Surface Bacteria Are Indirectly Associated with Oviposition Aversion in Bactrocera dorsalis.

Finding a suitable oviposition site is a challenging task for a gravid female fly, because the hatched maggots have limited mobility, making it difficult to find an alternative host. The oriental fruit fly, Bactrocera dorsalis, oviposits on many types of fruits. Maggots hatching in a fruit that is already occupied by conspecific worms will face food competition. Here, we showed that maggot-occupied fruits deter B. dorsalis oviposition and that this deterrence is based on the increased ß-caryophyllene concentration in fruits. Using a combination of bacterial identification, volatile content quantification, and behavioral analyses, we demonstrated that the egg-surface bacteria of B. dorsalis, including Providencia sp. and Klebsiella sp., are responsible for this increase in the ß-caryophyllene contents of host fruits. Our research shows a type of tritrophic interaction between micro-organisms, insects, and insect hosts, which will provide considerable insight into the evolution of insect behavioral responses to volatile compounds.

Use of Onion (Allium cepa) and Garlic (Allium sativum) Wastes for the Prevention of Fungal Disease (Saprolegnia parasitica) on Eggs of Rainbow Trout (Oncorhynchus mykiss).

This study was designed to prevent fungal disease (Saprolegnia parasitica) that occurs on rainbow trout eggs (Oncorhynchus mykiss) by using wastes of onion (Allium cepa) and garlic (Allium sativum) plants. For this purpose, fertilized rainbow trout eggs were exposed to garlic skin, garlic stem and onion skin aqueous methanolic extracts by bathing in concentrations of 0.4, 0.8, 1.6 and 3.2 g/L, whereas the control group was left untreated. The larvae in all groups were monitored until they become free-swimming larvae, the number of eggs died due to fungus was recorded, and the data obtained from experimental groups and control group were compared. As a result, it was determined that onion skin had no effect on the number of eggs that died due to fungus (p > .05), while garlic skin and garlic stem extracts significantly reduced the number of fungal infestations without affecting the number of live larvae or the embryological development of the eggs (p < .05). In light of these data, we conclude that garlic skin (0.4, 0.8, and 1.6 g/L) and garlic stem (0.8 and 1.6 g/L) aqueous methanolic extracts are effective at preventing Saprolegnia parasitica infestation on rainbow trout eggs and may be used in aquaculture.

In the beginning: egg-microbe interactions and consequences for animal hosts.

Microorganisms are associated with the eggs of many animals. For some hosts, the egg serves as the ideal environment for the vertical transmission of beneficial symbionts between generations, while some bacteria use the egg to parasitize their hosts. In a number of animal groups, egg microbiomes often perform other essential functions. The eggs of aquatic and some terrestrial animals are especially susceptible to fouling and disease since they are exposed to high densities of microorganisms. To overcome this challenge, some hosts form beneficial associations with microorganisms, directly incorporating microbes and/or microbial products on or in their eggs to inhibit pathogens and biofouling. Other functional roles for egg-associated microbiomes are hypothesized to involve oxygen and nutrient acquisition. Although some egg-associated microbiomes are correlated with increased host fitness and are essential for successful development, the mechanisms that lead to such outcomes are often not well understood. This review article will discuss different functions of egg microbiomes and how these associations have influenced the biology and evolution of animal hosts. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Parental Care Alters the Egg Microbiome of Maritime Earwigs.

Recruitment of beneficial microbes to protect offspring, often reducing the energetic costs of care, is now recognized as an important component of parental care in many animals. Studies on earwigs (order Dermaptera) have revealed that removal of females from egg tending increases mortality of eggs due to fungal infections, possibly caused by changes in the bacterial microbiome on the egg surface. We used a controlled female-removal experiment to evaluate whether female nest attendance in the maritime earwig, Anisolabis maritima, influences the bacterial microbiome on the egg surface. Further, we analyzed the microbiomes of mothers and their eggs to determine if there are a core set of bacteria transferred to eggs through female care. Microbiomes were analyzed using 16S rRNA bacterial DNA sequencing, revealing that bacterial operational taxonomic unit (OTU) richness and diversity were both significantly higher for female attended versus unattended eggs. The core microbiome of adult females contained bacteria which have the potential to carry anti-fungal characteristics; these bacteria were found in higher presence and relative abundance on eggs where females were allowed to provide care. These results demonstrate that female egg attendance significantly impacts the bacterial microbiome of A. maritima eggs, and identifies specific bacteria within the egg microbiome that should be investigated further for beneficial anti-fungal properties in this system.

Wolbachia (Rickettsiales: Alphaproteobacteria) Infection in the Leafhopper Vector of Sugarcane White Leaf Disease.

Wolbachia is a maternally inherited bacterium ubiquitous in insects that has attracted interest as a prospective insect pest-control agent. Here, we detected and characterized Wolbachia in the leafhoppers Matsumuratettix hiroglyphicus (Matsumura) (Cicadellidae: Hemiptera) and Yamatotettix flavovittatus Matsumura (Cicadellidae: Hemiptera), insect vectors of the phytoplasma that cause white leaf disease in sugarcane. The 16S rRNA and wsp gene markers revealed that Wolbachia was not present in the M. hiroglyphicus but naturally occurs in Y. flavovittatus. Additionally, the infection rates in adult leafhoppers ranged from 0 to 100% depending on geographic location. Moreover, Wolbachia was detected in the eggs and first- to fifth-instar nymphs of Y. flavovittatus. A phylogenic tree of Wolbachia indicated that it resided in the monophyletic supergroup B clade and clustered in the Ori subgroup. Furthermore, fluorescence in situ hybridization revealed that Wolbachia localized to the egg apices, randomly distributed in the egg cytoplasm, and was concentrated in the nymph and adult bacteriomes, as well as occasional detection in the thorax and abdomen. To the best of our knowledge, the present study is the first to demonstrate the prevalence of Wolbachia in the leafhopper Y. flavovittatus. The obtained results would provide useful information for the future development of Wolbachia as a biological control agent for the leafhopper vectors.

Use of bacteriophage vB_Pd_PDCC-1 as biological control agent of Photobacterium damselae subsp. damselae during hatching of longfin yellowtail (Seriola rivoliana) eggs.

AIMS: This study describes the effect of phage therapy on hatching of longfin yellowtail (Seriola rivoliana) eggs challenged with Photobacterium damselae subsp. damselae. METHODS AND RESULTS: A lytic phage (vB_Pd_PDCC-1) against P. damselae subsp. damselae was isolated and characterized. The use of phage vB_Pd_PDCC-1 increased the hatching rate of eggs, and reduced presumptive Vibrio species to non-detectable numbers, even in non-disinfected eggs. High-throughput 16S rRNA gene sequencing analysis revealed that phage vB_Pd_PDCC-1 caused significant changes in the composition and structure of the associated microbiota, allowing that members (e.g. those belonging to the family Vibrionaceae) of the class Gammaproteobacteria to be displaced by members of the class Alphaproteobacteria. CONCLUSIONS: To the best of our knowledge, this represents the first study evaluating phage therapy to control potential negative effects of P. damselae subsp. damselae during hatching of longfin yellowtail eggs. SIGNIFICANCE AND IMPACT OF THE STUDY: The Seriola genus includes several important commercial fish species due to its rapid growth and easy adaptability to confinement conditions. However, bacterial infections (especially those caused by Vibrio and Photobacterium species) are among the main limiting factors for the intensification of marine fish aquaculture, particularly during early development stages. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

Use of reactive oxygen species (ozone, hydrogen peroxide) for disinfection of hatching eggs.

The sample consisted of 480 hatching eggs of Japanese quails and was divided into 4 groups. Before the transfer to the incubator, the first group was not disinfected (negative control). In the second group, eggs were disinfected by means of formaldehyde fumigation (positive control). In the third and fourth group, reactive oxygen forms were used for disinfection- perhydrol (H2O2) and ozone (O3), respectively. Eggs were incubated under standard conditions. On the 14th D, eggs were candled, and proportions of fertilized eggs and died embryos were calculated. In addition, samples were collected for microbiological examination. After 17.5 D, the results of the whole hatching were evaluated. Chicks were reared for 14 D. Their survivability and body weight gain were recorded. Disinfection by means of reactive oxygen forms did not prove to be more effective in reducing the number of bacterial colonies on the shell. Reduced hatching and significantly increased mortality in the O3 group may indicate the negative impact of this gas on developing embryos. The results of hatching from eggs disinfected with H2O2 did not differ from those obtained in control groups. The biggest chicks were obtained from O3 disinfected eggs. However, during rearing, their growth did not match the one observed for birds in the remaining groups. Chicks hatched from eggs disinfected with H2O2 were characterized by the largest survivability. Disinfection with reactive oxygen forms did not significantly improve the hygiene of hatching eggs, hatching performance, and quality of hatched chicks. Hydrogen peroxide, whose application offered satisfactory hatching results, may be the recommended disinfectant. On the other hand, O3 appears to be undesirable because of its negative impact on bird embryos.

In ovo inoculation of duck embryos with different strains of Bacillus cereus to analyse their synergistic post-hatch anti-allergic potentialities.

BACKGROUND: Bacillus cereus is a Gram-positive, facultative anaerobic bacteria with few strains reported to be used as probiotics for animals and birds in recent times if the doses are formulated properly. OBJECTIVES: To analyse the synergistic anti-allergic potentiality of different Bacillus cereus strains on experimental in ovo and in vitro duck model, as probiotic immune stimulant. MATERIALS AND METHODS: Different strains of Bacillus cereus from 29 isolates were identified through 16S rRNA gene sequencing from the milk samples of buffalo breeds of South Asia. The probiotic properties were tested in aspects of gram staining, catalase test, coagulase, test, bile salt tolerance, pH tolerance and phenol tolerance test. MIC50 and MIC90 levels were profiled using nine different antibiotics, and antimicrobial activity against eight different enteric pathogens was assessed. Finally, the test strains of Bacillus cereus (Colony Forming Unit [CFU] 30X1011 ) were combined-infused at different concentrations in embryonated duck eggs to assess the post-hatch anti-allergic effects against histamine-induced allergic reaction and their immunoglobulin E (IgE) level was tested. RESULTS: Molecular identification confirmed the test strains as B. cereus HKS 1-1, B. cereus LOCK 1,002 and B. cereus BF2, which were all motile, spore-forming, catalase-positive and rod-shaped. All were 0.3% bile salt, 0.4% phenol and pH tolerant. Two-way ANOVA test P values revealed that B. cereus BF2 was statistically significant (p < .0014) in bile salt tolerance test. B. cereus HKS 1-1 was significant in phenol and pH tolerance at p < .0002 and p < .0489, respectively. Besides, the test strains showed antibiotic sensitivity and antimicrobial activity to different enteric pathogens. In vivo model referred the test strains as effective in partial allergy reduction at same CFU but at different concentrations with p < .0001 among the groups. CONCLUSION: The isolated and characterized strains of B. cereus showed partial immune-stimulating potentiality against experimentally induced allergic reaction.