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Invasion of Wolbachia into Anopheles and other insect germlines in an ex vivo organ culture system.

ABSTRACT: The common bacterial endosymbiont Wolbachia manipulates its host's reproduction to promote its own maternal transmission, and can interfere with pathogen development in many insects making it an attractive agent for the control of arthropod-borne disease. However, many important species, including Anopheles mosquitoes, are uninfected. Wolbachia can be artificially transferred between insects in the laboratory but this can be a laborious and sometimes fruitless process. We used a simple ex vivo culturing technique to assess the suitability of Wolbachia-host germline associations. Wolbachia infects the dissected germline tissue of multiple insect species when the host tissue and bacteria are cultured together. Ovary and testis infection occurs in a density-dependent manner. Wolbachia strains are more capable of invading the germline of their native or closely related rather than divergent hosts. The ability of Wolbachia to associate with the germline of novel hosts is crucial for the development of stably-transinfected insect lines. Rapid assessment of the suitability of a strain-host combination prior to transinfection may dictate use of a particular Wolbachia strain. Furthermore, the cultured germline tissues of two major Anopheline vectors of Plasmodium parasites are susceptible to Wolbachia infection. This finding further enhances the prospect of using Wolbachia for the biological control of malaria.

SUBMITTER: Hughes GL

PROVIDER: S-EPMC3340357 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Invasion of Wolbachia into Anopheles and other insect germlines in an ex vivo organ culture system.

Hughes Grant L GL Pike Andrew D AD Xue Ping P Rasgon Jason L JL

PloS one 20120430 4

The common bacterial endosymbiont Wolbachia manipulates its host's reproduction to promote its own maternal transmission, and can interfere with pathogen development in many insects making it an attractive agent for the control of arthropod-borne disease. However, many important species, including Anopheles mosquitoes, are uninfected. Wolbachia can be artificially transferred between insects in the laboratory but this can be a laborious and sometimes fruitless process. We used a simple ex vivo c ...[more]

PMID: 22558418

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Project description:BACKGROUND:Fetal alcohol spectrum disorder (FASD) is the leading nongenetic cause of mental retardation. There are no treatments for FASD to date. Preclinical in vivo and in vitro studies could help in identifying novel drug targets as for other diseases. Here, we describe an ex vivo model that combines the physiological advantages of prenatal ethanol (EtOH) exposure in vivo with the uniformity of primary fetal hippocampal culture to characterize the effects of prenatal EtOH. The insulin signaling pathways are known to be involved in hippocampal functions. Therefore, we compared the expression of insulin signaling pathway genes between fetal hippocampi (in vivo) and primary hippocampal culture (ex vivo). The similarity of prenatal EtOH effects in these 2 paradigms would deem the ex vivo culture acceptable to screen possible treatments for FASD. METHODS:Pregnant Sprague-Dawley rats received 1 of 3 diets: ad libitum standard laboratory chow (control-C), isocaloric pair-fed (nutritional control), and EtOH containing liquid diets from gestational day (GD) 8. Fetal male and female hippocampi were collected either on GD21 (in vivo) or on GD18 for primary culture (ex vivo). Transcript levels of Igf2, Igf2r, Insr, Grb10, Rasgrf1, and Zac1 were measured by reverse transcription quantitative polymerase chain reaction. RESULTS:Hippocampal transcript levels differed by prenatal treatment in both males and females with sex differences observed in the expression of Igf2 and Insr. The effect of prenatal EtOH on the hippocampal expression of the insulin pathway genes was parallel in the in vivo and the ex vivo conditions. CONCLUSIONS:The similarity of gene expression changes in response to prenatal EtOH between the in vivo and the ex vivo conditions ascertains that these effects are already set in the fetal hippocampus at GD18. This strengthens the feasibility of the ex vivo primary hippocampal culture as a tool to test and screen candidate drug targets for FASD.

Project description:BackgroundThe development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures allow for preservation of the native IVD structure, and adjacent vertebral bodies facilitate the application and control of mechanical loads. The purpose of this study was to establish loading and organ culture methods for rabbit IVD motion segments to study the effect of static load on the whole disc organ.MethodsIVD motion segments were harvested from rabbit lumbar spines and cultured in no-loading 6-well plates (control conditions) or custom-made apparatuses under a constant, compressive load (3 kg, 0.5 MPa) for up to 14 days. Tissue integrity, matrix synthesis, and the matrix gene expression profile were assessed after 3, 7, and 14 days of culturing and compared with those of fresh tissues.ResultsThe results showed that ex vivo culturing of motion segments preserved tissue integrity under no-loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days. Proteoglycan contents were decreased under both conditions, with a more obvious decrease under static load, and proteoglycan gene expression was also downregulated. However, under static load, immunohistochemical staining intensity and collagen Type II alpha 1 (COL2A1) gene expression were significantly enhanced (61.54 ± 5.91, P = 0.035) and upregulated (1.195 ± 0.040, P = 0.000), respectively, compared with those in the controls (P < 0.05). In contrast, under constant compression, these trends were reversed. Our initial results indicated that short-term static load stimulated the synthesis of collagen Type II alpha 1; however, sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content.ConclusionsA loading and organ culture system for ex vivo rabbit IVD motion segments was developed. Using this system, we were able to study the effects of mechanical stimulation on the biology of IVDs, as well as the pathomechanics of IVD degeneration.

Project description:BackgroundEx vivo disc organ culture systems have become a valuable tool for the development and pre-clinical testing of potential intervertebral disc (IVD) regeneration strategies. Bovine caudal discs have been widely selected due to their large availability and comparability to human IVDs in terms of size and biochemical composition. However, despite their extensive use, it remains to be elucidated whether their nutrient microenvironment is comparable to human degeneration.AimsThis work aims to create the first experimentally validated in silico model which can be used to predict and characterize the metabolite concentrations within ex vivo culture systems.Materials & methodsFinite element models of cultured discs governed by previously established coupled reaction-diffusion equations were created using COMSOL Multiphysics. Experimental validation was performed by measuring oxygen, glucose and pH levels within discs cultured for 7 days, in a static compression bioreactor.ResultsThe in silico model was successfully validated through good agreement between the predicted and experimentally measured concentrations. For an ex vivo organ cultured in high glucose medium (4.5 g/L or 25 mM) and normoxia, a larger bovine caudal disc (Cd1-2 to Cd3-4) had a central concentration of ~2.6 %O2, ~8 mM of glucose and a pH value of 6.7, while the smallest caudal discs investigated (Cd6-7 and Cd7-8), had a central concentration of ~6.5 %O2, ~12 mM of glucose and a pH value of 6.9.DiscussionThis work advances the knowledge of ex vivo disc culture microenvironments and highlights a critical need for optimization and standardization of culturing conditions.ConclusionUltimately, for assessment of cell-based therapies and successful clinical translation based on nutritional demands, it is imperative that the critical metabolite values within organ cultures (minimum glucose, oxygen and pH values) are physiologically relevant and comparable to the stages of human degeneration.

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Invasion of Wolbachia into Anopheles and other insect germlines in an ex vivo organ culture system.

Publications

Invasion of Wolbachia into Anopheles and other insect germlines in an ex vivo organ culture system.

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