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A yellow-head-like virus from Penaeus monodon cultured in Australia
Abstract and Figures
A rod-shaped, gill-associated virus (GAV) was found to be associated with mortalities of cultured adult Penaeus monodon from 4 farms in Queensland, Australia, from January to June 1996. infected prawns were observed swimming at the surface and edges of ponds and displayed varying degrees of red body colouration. The lymphoid organs of infected prawns appeared disorganized, were devoid of normal tubule structure and contained highly eosinophilic areas which corresponded to foci of highly infected and necrotic cells. Rod-shaped, enveloped virions and helical nucleocapsids were identified in lymphoid organ and gill cells. Nucleocapsids were 166-435 nm x 16-18 nm and enveloped virions were 183-200 nm x 34-42 nm. GAV isolated from infected prawns collected from 3 farms was successfully transmitted to healthy adult P. monodon, resulting in mortality from 7 to 8 d postinoculation. Mortality also occurred in prawns infected experimentally with GAV derived from lymphoid organs and gills. The morphology and cytopathology of GAV closely resemble that observed for lymphoid organ virus (LOV) from Australia and yellow-head virus (YHV) from Thailand. Molecular data are required to determine the phylogenetic relationships and appropriate taxonomic classification of these 3 prawn viruses.
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... The NCBI database contains sequences listed as GAV collected from Australia, Indonesia, Malaysia, Thailand and Taiwan (https://www.ncbi.nlm.nih.gov/, accessed 9/08/21), however, disease events in P. monodon production have only been reported from Australian sourced samples (Spann et al., 1997). GAV was associated with mortality events termed Mid-Crop Mortality syndrome (MCMS) that occurred in Australian P. monodon pond systems during 1996/97 (Anderson and Owens, 2001). ...
... GAV was associated with mortality events termed Mid-Crop Mortality syndrome (MCMS) that occurred in Australian P. monodon pond systems during 1996/97 (Anderson and Owens, 2001). GAV particles in viral extracts from these disease events were observed under TEM (Spann et al., 1997). Although experimental challenges have been conducted for GAV in P. monodon (Spann et al., 1997;Noble et al., 2017;Noble et al., 2020), as with many historical reports, the challenge inoculum was not a purified viral extract and comprehensive screening for other pathogens in the viral inoculum is not widely reported. ...
... GAV particles in viral extracts from these disease events were observed under TEM (Spann et al., 1997). Although experimental challenges have been conducted for GAV in P. monodon (Spann et al., 1997;Noble et al., 2017;Noble et al., 2020), as with many historical reports, the challenge inoculum was not a purified viral extract and comprehensive screening for other pathogens in the viral inoculum is not widely reported. The findings of the present study suggest that the majority of wild-sourced shrimp positive for the detection of GAV have co-detections of one or more other pathogen targets (50.2%) including IHHNV, YHV-7 and When-2. ...
Disease presents a considerable challenge to the sustainability and development of global shrimp aquaculture. The Australian black tiger shrimp (Penaeus monodon) aquaculture industry is heavily reliant on wild-sourced broodstock for seedstock production, representing a large and under evaluated biosecurity risk. Currently, there is a paucity of quantitative, large-scale data detailing the extent of pathogenic agents in wild-sourced P. monodon broodstock. This study presents a comprehensive investigation of the presence and level of detection of endemic pathogen targets in wild-sourced P. monodon broodstock collected from the two primary sources of supply for the Australian shrimp aquaculture industry using quantitative polymerase chain reaction (qPCR). Broodstock pleopod samples (N = 7472) were analysed by TaqMan qPCR for the detection of six viral pathogens. In total, 44.9% of wild-sourced broodstock were positive for the detection of at least one pathogen target. Whitespot virus and Yellowhead virus-1 were not detected in any sample. Infectious hypodermal haematopoietic necrosis virus (30%) and Gill-associated virus (28.1%) were the most prevalent pathogen targets detected. Whenzhou (syn. Wenzhou) shrimp virus-2 and Yellowhead virus-7 were each cumulatively detected in less than 3% of broodstock samples. Geographic source, sex and year of collection of broodstock significantly influenced prevalence of detection. The current study will be valuable to the Australian shrimp aquaculture industry to improve understanding of the presence of pathogens in wild-sourced broodstock, along with informing management decisions related to wild broodstock collection and associated biosecurity practices.
... Unlike the research on the viral diseases of farmed crustaceans, the reports on the spread of wild crustacean viruses and coinfection with multi-pathogen are still very limited [7]. Spann et al. [57] reported that coinfection with MoV and GAV is very common in diseased P. monodon. Notably, YHV-8 and GAV belong to Okavirus, and MoV and OWV1 belong to Wenrivirus. ...
At present, there are few studies on the epidemiology of diseases in wild Chinese white shrimp Penaeus chinensis. In order to enrich the epidemiological information of the World Organisation for Animal Health (WOAH)-listed and emerging diseases in wild P. chinensis, we collected a total of 37 wild P. chinensis from the Yellow Sea in the past three years and carried out molecular detection tests for eleven shrimp pathogens. The results showed that infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), yellow head virus genotype 8 (YHV-8), and oriental wenrivirus 1 (OWV1) could be detected in collected wild P. chinensis. Among them, the coexistence of IHHNV and DIV1 was confirmed using qPCR, PCR, and sequence analysis with pooled samples. The infection with YHV-8 and OWV1 in shrimp was studied using molecular diagnosis, phylogenetic analysis, and transmission electron microscopy. It is worth highlighting that this study revealed the high prevalence of coinfection with YHV-8 and OWV1 in wild P. chinensis populations and the transmission risk of these viruses between the wild and farmed P. chinensis populations. This study enriches the epidemiological information of WOAH-listed and emerging diseases in wild P. chinensis in the Yellow Sea and raises concerns about biosecurity issues related to wild shrimp resources.
... The virus has been divided into 8 subtypes [1][2][3][4], and it has been shown that only Type-1 (YHV-1) and Type-8 (YHV-8) [4] cause rapid and severe mortality. This contrasts with 6 less virulent variants (Types 2 to 7), one of which (YHV-2) has been given the specific name of gill associated virus (GAV) [5,6]. Penaeus (Penaeus) monodon and Penaeus (Litopenaeus) vannamei (the two main shrimp species cultivated in Thailand) are both highly susceptible to YHD caused by two known variants of YHV-1 (YHV-1a and -1b) [7,8]. ...
This short paper on yellow head virus Type-1 (YHV-1) of shrimp describes preliminary research on the potential for using YHV-1 attenuated in insect cells to protect shrimp against yellow head disease (YHD). YHV-1 can cause severe mortality in the cultivated shrimp Penaeus (Penaeus) monodon and Penaeus (Litopenaeus) vannamei. No practical vaccination has been reported. The C6/36 mosquito cell cultures inoculated with YHV-1 become positive by PCR and by immunocytochemistry (immunopositive) for up to 30 split-cell passages. Shrimp injected with homogenates from low-passage cultures die from typical YHV-1 disease while shrimp injected with homogenates from high passage cultures do not, even though they become PCR positive and immunopositive for YHV-1. This suggested that viral attenuation had occurred during insect-cell passaging, and it opened the possibility of using homogenates from high-passage insect cultures as a vaccine against YHV-1. To test this hypothesis, homogenates from 30th-passage, YHV-positive cultures were injected into shrimp followed by challenge with virulent YHV-1. Controls were injected with homogenate from 30th-passage, naive (normal stock) insect-cell cultures. No shrimp mortality occurred following injection of either homogenate, but shrimp injected with the YHV-1 homogenate became both RT-PCR positive and immunopositive. Upon challenge 10 days later with YHV-1, mortality in shrimp injected with naive insect-cell homogenate was 100% within 7 days post-challenge while 100 % mortality in the YHV-1 homogenate group did not occur until day 9 post-challenge. Kaplan-Meier log-rank survival analysis revealed that survival curves for the two groups were significantly different (p<0.001). The cause of delay in mortality may be worthy of further investigation.
... merguiensis) [3], and can stunt growth at juvenile stages in growout ponds for both Japanese tiger prawn (P. japonicas) [4] and farmed black tiger shrimp (P. monodon) [5,6]. ...
Background
Viral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming. HPV virus titre is commonly determined by quantitative PCR (qPCR), which is a time-consuming method requiring laboratory equipment unsuitable for field implementation. The aim of this study was to develop a simple, robust, rapid and reliable method to detect HPV in low-resource environments.
Methods
We developed a rapid shrimp HPV test that uses (1) a simple three-step sample preparation protocol, followed by (2) isothermal recombinase polymerase amplification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp sample matrix, and retrospective testing of Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity.
Results
The rapid shrimp HPV test could detect as little as 35 genome-equivalent copies per reaction in homogenized F . merguiensis banana shrimp. Retrospective testing of stored tissues (n = 33) indicated 100% diagnostic sensitivity (95% confidence interval, CI: 86–100%) and 100% specificity (95% CI: 66–100%) for detection of HPV.
Conclusion
The rapid shrimp HPV test could be completed in only 40 minutes, and required only homogenization pestles, some pipettors, and a small heating block for single temperature incubation at 39°C. Critically, our procedure eliminated the time-consuming purification of nucleic acids from samples and when combined with RPA-LFD offers a user-friendly HPV detection format that can potentially be performed on-site. Our approach represents a major step forward in the development of a simple and sensitive end-point method for quick determination of unfavourable HPV virus numbers in shrimp, and has great potential to advance on-site management of shrimps in aquaculture.
... 1,9 Apart from interactions, cannibalism can be documented as another dominant disease transmission route among the species. For example, Spawner-isolated fatality viruses 10 in Pandalusplatyceros and Gill-associated virus 11 in Penaeusmondon are transmitted through cannibalism among the population. Cannibalism is prominent and wide-spreading biological phenomenon eliminating and predating con-specifics in a prey-predator inter plays. ...
In this paper, we investigate a cannibalistic predator‐prey system with Beddington‐DeAngelis type response function when the infection only exists in the predator species. The predator species are subject to cannibalistic interaction and the infection spreads among the predators via cannibalism. The model is studied in presence of multiple delays for the maturation of prey and predator. Local stability analysis of the predator‐prey model around the biologically realistic two essential steady states is investigated. We also investigate the bifurcation analysis of the proposed model around the co‐existing steady states. Using the normal form method and center manifold theory, the direction of Hopf bifurcation and the Hopf bifurcating periodic solutions are explored. In presence of time delays, we derive the criteria for the permanence of the model. Our findings demonstrate that maturation delays destabilize the system and can produce high periodic oscillations. Cannibalism can act as a self‐regulatory scenario controlling the transmission of disease among the predator species and stabilizing the oscillations in the predator‐prey system. Our theoretical analysis is well supported with numerical simulation.
... Infection corresponds to a multifocal to generalised necrosis of connective tissues of the gill, heart, hepatopancreas and gut, with nuclei showing pyknosis and karyorrhexis. Enveloped rod-shaped virions measuring 60-110 x 24-42 nm were identified within the cytoplasm of affected cells, nonmature particles were occasionally associated with the endoplasmic reticulum, similar to that described by Spann et al. (1997) in GAV infections. Tissue tropism, histopathology and ultrastructural characteristics suggest this virus is similar to viruses within the family Roniviridae and genus Okavirus, such as YHV, GAV, however genome sequencing is needed to clarify this relationship. ...
Farming of crustaceans especially shrimp, crabs and crayfish have expanded significantly over the past four decades. The aquaculture production of crustaceans is now a multimillion-dollar industry providing jobs to millions of people around the world especially in countries with large coastal boundaries in Asia and Latin America. Crustacean farming is largely dominated by penaeid shrimp aquaculture. The emergence of infectious diseases especially diseases of viral origin has been a threat to this nascent industry. Many viruses that affect penaeid shrimp have been relatively well characterised due to their economic importance. These include viruses with single-stranded DNA containing genomes such as Infectious Hypodermal, and Hematopoietic Necrosis Virus, and Hepatopancreatic Parvovirus (family: Parvoviridae), double stranded DNA viruses such as white spot syndrome virus (family: Nimaviridae), Penaeus monodon nudivirus (Family: Nudiviridae), Decapod iridescent virus 1 (family: Iridoviridae), and Baculovirus penaei (tentatively classified in the family: Baculoviridae), single-stranded RNA viruses such as Taura syndrome virus (family: Dicistroviridae), yellow head, and Gill-associated viruses (family: Roniviridae), and Macrobrachium rosenbergii Nodavirus (family: Nodaviridae), and double-stranded RNA virus such as infectious myonecrosis virus (Totiviridae-like). White spot syndrome virus is of major concern as the virus has a wide host range and poses a threat to wild and farmed populations of decapod crustacean species, with multiple species showing differing levels of susceptibility. Viral infections have been reported in wild crustacean species including those which are commercially exploited. However, in comparison to cultured species relatively little is known about the effects of viruses in wild crustaceans.
... So this study can help in the identification of root of the coronavirus outbreak as still there are no reports for the source of this coronavirus which has recently emerged in 2019 and still is the major cause of death to humans as well as to some animals. It is hoped that this review will enable the discovery and identification of source of coronaviruses and facilitate further research into their molecular biology, phylogeny and evolution [25][26][27][28][29][30][31][32][33][34]. ...
Yellow head disease (YHD) is caused by yellow head virus genotype 1 (YHV-1), which is a positive-sense (+) ssRNA virus that belongs to the genus Okavirus, family Roniviridae, order Nidovirales. There are currently eight recognized genotypes. The virions are rod-shaped (bacilliform) and approximately 35–60 nm × 140–200 nm with rounded ends. YHD was first recognized in Thailand where outbreaks of YHD caused substantial economic losses in the 1990s, but losses decreased after the widespread introduction of specific pathogen-free (SPF) seedstocks of Penaeus vannamei around 2002. YHV genotype 8 (YHV-8), recently described from China also appears to have high virulence. The infection of YHV can be transmitted vertically and horizontally. Control of YHD is focused on the use of SPF stocks in biosecure culture settings.
Crustaceans are an important source of aquatic food protein and play a large role in securing global food security. Yet, despite the high economic value and importance of this product for global food security, disease is still the number one issue in limiting yields. Viral pathogens are known to exert significant constraints on the growth and survival of crustacea under culture conditions. However, in comparison to cultured species, relatively little is known about the effects of viruses in wild crustaceans. Unless farmed, mass mortalities of aquatic animals are rarely detected and studied; so, it is difficult to assess the impact viral infections can have upon a wild population. To date, there are at least 100 viral pathogens across multiple viral families (DNA and RNA) described within shrimp, crabs, crayfish, and lobsters. The majority of these viral infections have been tentatively assigned to viral families based upon structural characteristics due to lack of genomic data. However, this will become clearer and the field will continue to evolve as sequencing methods continue to develop, including recent virome studies, which have resulted in the identification of previously undescribed viral sequences. This chapter aims to provide an overview of viral infections of crustaceans, providing an overview of the main viral infections that have been described within crustacean species, highlighting the viral diversity present in both farmed and wild species.
A polychaete-assisted sand filter (PASF) can aid water recirculation systems for shrimp culture ponds by removing large amounts of nutrients and suspended solids and chemically treating wastewater. By ingesting and degrading organic matter, Perinereis helleri polychaetes reared in a PASF can accumulate and potentially remove infectious hypodermal and haemopoietic necrosis virus (IHHNV) present in wastewater from ponds rearing virus-infected Penaeus monodon shrimp. Reported here are data showing that filtering pond wastewater through a PASF reduces its potential to transmit IHHNV. The trial employed 3 groups of 4 tanks each containing 20 P. monodon randomly selected from a pond in which IHHNV was evident at moderate prevalence and low loads (IHHNV low-load Pond 2). Over a 2 week period, each tank group was supplied with wastewater from either the same pond (Pond 2) or Pond 1 in which IHHNV was 100% prevalent at ~10⁴-fold higher infection loads, either directly or after being filtered through a PASF. IHHNV real-time qPCR data on total nucleic acid (TNA) extracted from pleopods of 35 P. monodon selected at random from each group identified an elevated IHHNV infection prevalence (91%) in shrimp tested from tanks supplied directly with wastewater from Pond 1. In comparison, IHHNV was detected at a much reduced prevalence and lower loads among shrimp from tanks supplied with wastewater from Pond 2 (33%) or from Pond 1 after it had been filtered through a PASF (31%). The IHHNV prevalence and load data indicate that a PASF can play a useful role in reducing the potential for shrimp pond wastewater to transmit IHHNV infection to naïve P. monodon.
Yellow-head virus (YHV) causes acute infections in Penaeus monodon that result in very high mortality. First reports of the virus suggested that the viral core consisted of DNA and that the virus should be classified as a granulosis-type baculovirus. However, 3 attempts at DNA extraction with high concentrations of purified virus (verified by transmission electron microscopy, TEM) gave only traces of DNA, which could not be visualized by ethidium bromide staining of agarose electrophoresis gels. Although selected recombinant clones derived from these pooled DNA traces did not hybridize with host shrimp DNA, they also failed to react with YHV-infected tissue by the in situ DNA hybridization technique. Furthermore, negatively stained virions of YHV viewed by TEM were atypical for baculoviruses and viral assembly is cytoplasmic. Therefore, renewed attempts to extract nucleic acid from purified YHV preparations focused on RNA rather than DNA. Hemolymph was collected aseptically by syringe from 200 artificially YHV-infected, Live shrimp in terminal stages of the disease. Purified virions were prepared by a program of centrifugation culminating in 22% to 45% Urografin gradient ultracentrifugation. A band at the 30-37% interval of the gradient gave the cleanest preparation with the highest quantity of virions. By TEM these were enveloped, measured 150-170 x 40-50 nm and were surrounded by a fringe of knob-like projections approximately 11 nm in length. Nucleic acid was extracted using guanidium thiocyanate and purified by CsCl gradient ultracentrifugation. High-molecular-weight nucleic acid was obtained which was degraded by RNase-A but not by DNase I. Based on morphology of negatively stained virions by TEM and on RNA content, YHV resembles rhabdoviruses or coronaviruses, rather than baculoviruses. This is an important discovery, since it necessitates cDNA preparation in the process to develop a nucleic-acid probe for YHV detection by the in situ or dot blot hybridization techniques.
A recently reported disease syndrome of Penaeus monodon in Thailand is called 'yellow-head' or 'hua leung' in Thai. It is usually characterized by light yellow coloration of the dorsal cephalothorax area and generally pale or bleached appearance of affected prawns. The yellow color in the cephalothorax region results from the underlying yellow hepatopancreas showing through the translucent carapace in moribund shrimp. In histological preparations of moribund yellow-head specimens for the light microscope, no consistent bacterial, fungal or parasitic agents could be found. The lymphoid organs of yellow-head specimens showed extensive abnormalities. These included obviously necrotic cells and vacuolated cells with hypertrophied nuclei. Also evident were very densely basophilic, globose cytoplasmic inclusions located adjacent to some of the hypertrophied nuclei. Similar basophilic inclusions were found in interstitial hepatopancreatic tissue, in connective tissue underlying the mid gut, in cardiac tissue, in gill tissue and in hematopoetic tissue. Transmission electron micrographs revealed the presence of previously undescribed rod-shaped, enveloped virions in the cytoplasm adjacent to the nuclei of cells from various tissues. Free virions were also present in intercellular spaces. The virions were similar to those of the insect granulosis viruses (Baculoviridae) in terms of cytoplasmic location, size, morphology and development. However, they were not occluded by granulin.
Beginning in the spring of 1993, a serious mortality among cultured kuruma shrimp, Penaeus japonicus occurred in Japan. The typical sign of this disease was white spots on the inside surface of the carapace. Challenge tests demonstrated that the causative agent was highly virulent. This was demonstrated by injection of a filtrered homogenate of the lymphoid organ obtained from diseased shrimp. A non-occluded bacilliform virus was found by electron microscopy in the lymphoid organ of both naturally and experimentally infected shrimps. The virion was bacilliform and surrounded by a virion envelope (a lipid bilayer membrane). The dimension of the virion was 83 nm in diameter and 275 nm in length. From these results, the bacilliform virus is considered to be the causative agent of the disease.
A system for holding lobster hemocytes in a viable state was developed and utilized to study the in vitro phagocytosis of sheep red blood cells (SRBC) by lobster hemocytes.Using SRBC sensitized with the opsonin present in lobster serum, 2% of the hemocytes demonstrated phagocytosis detectable as early as 20 min after the hemocytes and erythrocytes were mixed and held at 15 C. Lower phagocytosis levels were observed with untreated erythrocytes. As with other poiklothermic animals, the temperature affected the rate of phagocytosis, which was first observed at the 60-min sampling in cells held at 4 C.
A new non-occluded baculo-like virus was detected from black tiger shrimp (Penaeus monodon) with yellow head disease in the southern part of Thailand. Virus particles observed in necrotic tissues of gills and lymphoid organ were bacilliform in shape, ranging from 150-200 nm in length and 40-50 nm in diameter, and were enveloped singly by a trilaminar unit membrane. From the experimental infection trials, it was assumed that this virus was the most virulent among the viruses which have been known in the black tiger shrimp, to date. Pathogenicity and some characters of the virus as well as pathological changes caused by this virus were described.
Infectivity and organ distribution studies for the rhabdovirus (rhabdovirus of penaeid shrimp-RPS) isolated from Penaeus spp. were conducted with subadult Penaeus stylirostris. Test shrimp were inoculated intramuscularly with different isolates of RPS from penaeid shrimp collected from different shrimp farms in Hawaii and Ecuador. Although the experimentally infected shrimp showed no clinical or gross manifestations of disease, the RPS was found to be infectious for the animals. Virus replication was demonstrated only in the lymphoid (Oka) organs of infected shrimp by virus assay and immunofluorescence. Also, the Oka organs of infected animals showed gross cellular changes and were significantly larger in size than the corresponding organs from uninfected shrimp. A possible role of the RPS in the causation of disease in penaeid shrimp is postulated.
Subadult Penaeus monodon shrimp were sampled from 4 farms in Queensland, Australia. Histological observations on lymphoid organs showed the formation of abnormal cell foci, which resembled tubules lacking a central hemolymph vessel. These formations contained hypertrophied nuclei with marginated chromatin, vacuolated cells and inclusion bodies which stained positive with Feulgen's reaction. Male and female P. monodon broodstock were sampled from 1 farm and showed the same abnormal histological features within the lymphoid organ as the subadults. Electron microscopy revealed tightly enveloped, cylindrical particles, 163-200 nm x 36-63 nm, packed into paracrystalline arrays within the cytoplasm and infrequently within the nucleus. Arrays of virions were also observed within the cytoplasm of broodstock gill cells. Cross-sections of the particles revealed electron dense nucleic acid cores and envelopes. Nucleocapsids, 83-590 nm x 13-15 nm, were seen free and in vesicles within the cytoplasm. A negatively stained preparation of partially purified lymphoid organ revealed a nucleocapsid with one conical end. Lymphoid organ virus (LOV) resembles yellow-head virus (YHV) from Thailand in its morphology and cytopathology and rhabdovirus of penaeid shrimp (RPS) from the Americas in its cytopathology.
A new virus was isolated from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps. The virus was isolated from two species of penaeid shrimps obtained from three different sources employing a previously developed cell-culture assay. Electron-microscopical studies of both purified virus and infected cells showed bullet-shaped particles identifying it as a rhabdovirus.
A low-viscosity embedding medium based on ERL-4206 is recommended for use in electron microscopy. The composition is: ERL-4206 (vinyl cyclohexene dioxide) 10 g, D.E.R. 736 (diglycidyl ether of polypropylene glycol) 6 g, NSA (nonenyl succinic anhydride) 26 g, and S-1 (dimethylaminoethanol or DMAE) 0.4 g. The medium is easily and rapidly prepared by dispensing the components, in turn by weight, into a single flask. The relatively low viscosity of the medium (60 cP) permits rapid mixing by shaking and swirling. The medium is infiltrated into specimens after the use of any one of several dehydrating fluids, such as ethanol, acetone, dioxan, hexylene glycol, isopropyl alcohol, propylene oxide, and tert.-butyl alcohol. It is compatible with each of these in all proportions. After infiltration the castings are polymerized at 70°C in 8 hours. Longer curing does not adversely affect the physical properties of the castings. Curing time can be reduced by increasing the temperature or the accelerator, S-1, or both; and the hardness of the castings is controlled by changes in the D.E.R. 736 flexibilizer. The medium has a long pot life of several days and infiltrates readily because of its low viscosity. The castings have good trimming and sectioning qualities. The embedding matrix of the sections is very resistant to oxidation by KMnO4 and Ba(MnO4)2, compared with resins containing NADIC methyl anhydride. Sections are tough under the electron beam and may be used without a supporting membrane on the grids. The background plastic in the sections shows no perceptible substructure at magnifications commonly used for biological materials. The medium has been used successfully with a wide range of specimens, including endosperms with a high lipid content, tissues with hard, lignified cell walls, and highly vacuolated parenchymatous tissues of ripe fruits.