Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea ariakensis Gould

Moribund specimens of the oyster, Crassostrea ariakensis Gould, aged 2-3 years were collected from Hailing Bay in Yangxi County of Guangdong Province from February to May and November to December in the years 2001, 2002, and 2003. A massive infection by an obligate intracellular prokaryote, specifically a rickettsia-like organism (RLO), was found. Here we report investigations of this RLO in the tissues of the oyster C. ariakensis Gould and describe the histology, ultrastructure, and morphogenesis of this pathogen in C. ariakensis Gould. Light microscopic observations of stained tissues revealed cytoplasmic inclusion bodies typical of prokaryote infection in about 87% (26/30) of the oysters. Most inclusions were observed in epithelial cells and connective tissues of the gill, mantle, and digestive gland of most of the infected oysters. The shape, size, and color of inclusions from different tissues were polymorphic. Electron microscopic examination of digestive gland, gill, and mantle tissues showed that the RLOs were intracytoplasmic. RLOs were often round, dumb-bell-shaped (undergoing binary fission), or occasionally rod-shaped and ranged from approximately 0.58 to 1.20microm in size. The organisms exhibited an ultrastructure characteristic of prokaryotic bacteria-like cells, including a trilaminar cell wall, electron-dense periplasmic ribosome zone, and a DNA nucleoid. Reproductive stages, including transverse binary fission, were observed by TEM. These stages were frequently observed within membrane-bound cytoplasmic vacuoles. Hexagonal phage-like particles in the cytoplasm of RLOs were also observed.     

Purification and antigenic characteristics of a rickettsia-like organism from the oyster Crassostrea ariakensis

A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants.     

Characterisation of an emerging rickettsia-like organism in Tasmanian farmed Atlantic salmon Salmo salar

A rickettsia-like organism (RLO) was observed in farmed Atlantic salmon Salmo salar located in south-east Tasmania, Australia. Several assays such as immunoperoxidase, immunoelectron microscopy, polymerase chain reaction and nucleic acid sequencing, as well as phylogenetic analysis of rDNA sequences, were performed on infected fish tissues. Immunohistochemistry results suggested the presence of related antigenic determinants between the Tasmanian RLO and the type strain LF-89 of Piscirickettsia salmonis. However, sequence alignment demonstrated that the Tasmanian RLO contains a 19 bp deletion at the 3'-end of the internal transcribed spacer region of the rDNA operon, indicating a genetic divergence from P. salmonis isolates, which are exotic to Australia.     

Ultrastructure and cytopathology of a rickettsia-like organism causing systemic infection in the redclaw crayfish, Cherax quadricarinatus (Crustacea: decapoda), in Ecuador

A study of the ultrastructural characteristics of an intracellular bacterium infecting the redclaw crayfish, Cherax quadricarinatus, a pathogen referred to previously as a rickettsia-like organism (RLO), revealed the presence of different developmental stages. These included a rod-shaped and uniformly electron-dense elementary body (EB) and an intermediate body (IB). The length of the EB varied between 0.48 and 0.6 microm, and the diameter was 0.3 microm. The IB was 0.75 to 1.1 microm long by 0.36 to 0.44 microm in diameter. Although the EB of this bacterium has ultrastructural characteristics similar to those of Rickettsiella, no information is available regarding its genetic relationship to this genus, and the intracellular bacterium should continue to be referred to as a rickettsia-like organism. The hemocytes had different levels of infection, and the RLO proliferated inside these cells. The EB appeared to be free in the cytoplasm of infected hemocytes and other cells; however, this might be a fixation artifact. The EB was also contained in membrane-bound vacuoles along with the IB. RLO colonies were observed inside small granular cells. No large granular cells were observed in the sections examined; therefore, no data were obtained regarding infection of this type of hemocyte. The fixed phagocytes on the external side of the terminal hepatic arterioles had an activated interrupted layer containing RLO bacteria. Stem cells in the hematopoietic tissue were also infected, and some cells were apparently being released into circulation.     

Pathological and epidemiological observations on rickettsiosis in cultured sea bass (Dicentrarchus labrax L.) from Greece

A systemic infection of a Rickettsia‐like organism (RLO) in cultured sea bass is described for the first time. In hatcheries, clinical signs were lethargy, inappetence and discoloration. Twenty days after transfer to sea cages from hatcheries where the disease existed, fish showed erratic and abnormal swimming behaviour, loss of orientation, and lethargy. Cumulative mortality in colder months of the year reached 30% in hatcheries and 80% in cages. Surviving fish in cages did not show any clinical signs of RLO infection in the subsequent year. Evidence for a systemic distribution of RLO was supported by histolopathological lesions in both infected hatchery and caged fish, where the lesion profile included cranial sensory, central nervous, integumental and alimentary organ systems. Intracranial lesions were primarily characterized by an ascending histiocytic perineuritis and necrotizing congestive meningoencephalitis, with evidence for transfer of infective agents across the blood–brain barrier confirmed by the presence of RLOs within capillary endothelium and histiocytes in inflamed regions of the optic tectum and the cerebellum. In the most severe cases, infection spread to the statoacoustical (semicircular) canal system and the ependymal lining of ventricles, with marked rickettsial‐laden histiocytic infiltration of the canal lumen. Integumental lesions were restricted to the oral submucosa, nares and integumental dermis of the cranium. Alimentary lesions were noted in both the liver parenchyma and mucosa/submucosa of the stomach. In all affected organs the RLOs were found by immunohistochemistry to be related to Piscirickettsia salmonis.     

HMGB in Mollusk Crassostrea ariakensis Gould: Structure,Pro-Inflammatory Cytokine Function Characterization and Anti-Infection Role of Its Antibody

Background

Crassostrea ariakensis Gould is a representative bivalve species and an economically important oyster in China, but suffers severe mortalities in recent years that are caused by rickettsia-like organism (RLO). Prevention and control of this disease is a priority for the development of oyster aquaculture. It has been proven that mammalian HMGB (high mobility group box) can be released extracellularly and acts as an important pro-inflammatory cytokine and late mediator of inflammatory reactions. In vertebrates, HMGB’s antibody (anti-HMGB) has been shown to confer significant protection against certain local and systemic inflammatory diseases. Therefore, we investigated the functions of Ca-HMGB (oyster HMGB) and anti-CaHMGB (Ca-HMGB’s antibody) in oyster RLO/LPS (RLO or LPS)-induced disease or inflammation.

Methodology/Principal Findings

Sequencing analysis revealed Ca-HMGB shares conserved structures with mammalians. Tissue-specific expression indicates that Ca-HMGB has higher relative expression in hemocytes. Significant continuous up-regulation of Ca-HMGB was detected when the hemocytes were stimulated with RLO/LPS. Recombinant Ca-HMGB protein significantly up-regulated the expression levels of some cytokines. Indirect immunofluorescence study revealed that Ca-HMGB localized both in the hemocyte nucleus and cytoplasm before RLO challenge, but mainly in the cytoplasm 12 h after challenge. Western blot analysis demonstrated Ca-HMGB was released extracellularly 4–12 h after RLO challenge. Anti-CaHMGB was added to the RLO/LPS-challenged hemocyte monolayer and real-time RT-PCR showed that administration of anti-CaHMGB dramatically reduced the rate of RLO/LPS-induced up-regulation of LITAF at 4–12 h after treatment. Flow cytometry analysis indicated that administration of anti-CaHMGB reduced RLO/LPS-induced hemocyte apoptosis and necrosis rates.

Conclusions/Significance

Ca-HMGB can be released extracellularly and its subcellular localization varies when stimulated with RLO. Ca-HMGB is involved in oyster immune reactions and functions as a pro-inflammatory cytokine. Anti-CaHMGB can significantly suppress RLO/LPS-induced inflammatory responses and hemocyte necrosis and apoptosis, suggesting that Ca-HMGB is a potential target to prevent and control RLO/LPS-induced disease or inflammation.     

Putative Phage Hyperparasite in the Rickettsial Pathogen of Abalone, “Candidatus Xenohaliotis californiensis”

Studies on the ecology of microbial parasites and their hosts are predicated on understanding the assemblage of and relationship among the species present. Changes in organismal morphology and physiology can have profound effects on host–parasite interactions and associated microbial community structure. The marine rickettsial organism, “Candidatus Xenohaliotis californiensis” (WS-RLO), that causes withering syndrome of abalones has had a consistent morphology based on light and electron microscopy. However, a morphological variant of the WS-RLO has recently been observed infecting red abalone from California. We used light and electron microscopy, in situ hybridization and16S rDNA sequence analysis to compare the WS-RLO and the morphologically distinct RLO variant (RLOv). The WS-RLO forms oblong inclusions within the abalone posterior esophagus (PE) and digestive gland (DG) tissues that contain small rod-shaped bacteria; individual bacteria within the light purple inclusions upon hematoxylin and eosin staining cannot be discerned by light microscopy. Like the WS-RLO, the RLOv forms oblong inclusions in the PE and DG but contain large, pleomorphic bacteria that stain dark navy blue with hematoxylin and eosin. Transmission electron microscopy (TEM) examination revealed that the large pleomorphic bacteria within RLOv inclusions were infected with a spherical to icosahedral-shaped putative phage hyperparasite. TEM also revealed the presence of rod-shaped bacteria along the periphery of the RLOv inclusions that were morphologically indistinguishable from the WS-RLO. Binding of the WS-RLO-specific in situ hybridization probe to the RLOv inclusions demonstrated sequence similarity between these RLOs. In addition, sequence analysis revealed 98.9–99.4 % similarity between 16S rDNA sequences of the WS-RLO and RLOv. Collectively, these data suggest that both of these RLOs infecting California abalone are “Candidatus Xenohaliotis californiensis,” and that the novel variant is infected by a putative phage hyperparasite that induced morphological variation of its RLO host.     

Tsetse immunity and the transmission of trypanosomiasis

Cyclical transmission of African trypanosomes - Trypanosoma congolense and subspecies of T. brucei - depends on their uptake by and development within their tsetse fly vectors. Tsetse susceptibility to such trypanosome infection seems to be controlled by maternally inherited rickettsia-like organisms (RLOs) (Fig. 1) and it now seems that the RLOs may exert this effect by controlling midgut lectins in the fly. Ian Maudlin and Susan Welburn explain the latest findings.     

类立克次体侵染中华绒螯蟹神经组织的光镜和电镜观察

对患颤抖病的中华绒螯解（Eriocheir sinensis）神经和肌肉组织进行光镜和电镜观察,结果显示类立克次体广泛存在于胸神经节中的神经胶质细胞、结缔组织以及细胞间隙中。在神经细胞与步足肌肉细胞连接处,即运动终板部位有大量类立克次体侵染,并且侵染方式与在胸神经节中一样,均以聚集成团的形式存在。胸神经节是支配步足的神经丛,而颤抖病是以步足颤抖为病症的疾病。类立克次体大量侵染胸神经节和运动终板部位,表明该微生物与病蟹步足颤抖的病症有密切关系,进一步证明类立克次体是颤抖病的致病微生物。     

Allograft Inflammatory Factor 1 Functions as a Pro-Inflammatory Cytokine in the Oyster,Crassostrea ariakensis

The oyster Crassostrea ariakensis is an economically important bivalve species in China, unfortunately it has suffered severe mortalities in recent years caused by rickettsia-like organism (RLO) infection. Prevention and control of this disease is a priority for the development of oyster aquaculture. Allograft inflammatory factor-1 (AIF-1) was identified as a modulator of the immune response during macrophage activation and a key gene in host immune defense reaction and inflammatory response. Therefore we investigated the functions of C. ariakensis AIF-1 (Ca-AIF1) and its antibody (anti-CaAIF1) in oyster RLO/LPS-induced disease and inflammation. Ca-AIF1 encodes a 149 amino acid protein containing two typical Ca²⁺ binding EF-hand motifs and shares a 48–95% amino acid sequence identity with other animal AIF-1s. Tissue-specific expression analysis indicates that Ca-AIF1 is highly expressed in hemocytes. Significant and continuous up-regulation of Ca-AIF1 is detected when hemocytes are stimulated with RLO/LPS (RLO or LPS). Treatment with recombinant Ca-AIF1 protein significantly up-regulates the expression levels of LITAF, MyD88 and TGFβ. When anti-CaAIF1 antibody is added to RLO/LPS-challenged hemocyte monolayers, a significant reduction of RLO/LPS-induced LITAF is observed at 1.5–12 h after treatment, suggesting that interference with Ca-AIF1 can suppress the inflammatory response. Furthermore, flow cytometric analysis indicated that anti-CaAIF1 administration reduces RLO/LPS-induced apoptosis and necrosis rates of hemocytes. Collectively these findings suggest that Ca-AIF1 functions as a pro-inflammatory cytokine in the oyster immune response and is a potential target for controlling RLO infection and LPS-induced inflammation.     

Rickettsia-Like Organism Causing Disease in a Crangonid Amphipod from Florida

A rickettsia-like organism causing high mortality and gross pathology has been found infecting over 40% of a population of the freshwater crustacean, Crangonyx (near) floridanus Bousfield, in Florida. This is the first report of a rickettsia-like organism from a strictly aquatic animal. Morphological and developmental stages of the organisms range in size and shape from small, dense bacilliform cells measuring 150 nm in diameter by 395 nm in length, to large sausage-shaped cells 0.5 by 3 mum.     

Studies on rickettsia-like organism disease of the tropical marine pearl oyster I: the fine structure and morphogenesis of pinctada maxima pathogen rickettsia-like organism

The present paper reports for the first time the discovery of a rickettsia-like organism (RLO) in the cultured tropical marine pearl oyster Pinctada maxima with mass mortality in the Hainan Province of China. This organism parasitizes the cytoplasm of host cells and forms intracytoplasmic eosinophilic inclusions. These organisms are extremely pleiomorphic in shape and average 967 x 551 nm in size, as measured in cross sections of transmission electron micrographs. The organisms exhibit clearly recognizable ultrastructural characteristics of prokaryotic bacteria-like cells, including two trilaminar membranes, an increasing electron-dense periplasmic ribosome zone, and a thread-like DNA nucleoidal structure. In addition to the above prokaryotic characteristics, the following unique biological characteristics were confirmed by TEM: (i) These organisms are usually located in host cells in two ways, namely, free in the cell cytoplasm and involved within membrane-limited phagolysosomes; (ii) The organisms exist in two morphological cell types, namely a small cell variant (SCV) and a large cell variant (LCV). The most important morphological difference between two cell types is that the SCV is obviously ribosome-rich in the periphery of the body, which makes SCV more electron-dense in the cytoplasm and narrower in the central nucleoid area than the LCV; (iii) Two propagative modes of the organisms, transverse binary fission and budding, are observed in cytoplasm and phagolysosomes of host cells under TEM, in which the budding is more often seen in phagolysosomes. These characteristics indicate that the organism is a separate species in the family Rickettsiaceae and should be classified into the genus Rickettsia. On the basis of the existence of the two propagative modes and two cell types, and intracellular location, we propose a developmental cycle for this organism which includes a vegetative differentiation stage to develop LCV by transverse binary fisson and a budding differentiation stage to develop resistant SCV. Copyright 1999 Academic Press.     

Occurrence and localization of spherical viruslike particles in tissues of apparently healthy tobacco thrips,Frankliniella fusca,a vector of tomato spotted wilt virus

Spherical viruslike particles (VLP) were found in the tissues of apparently healthy tobacco thrips, Frankliniella fusca. The particles occurred in abundance in thrips from Ontario but were absent in thrips from Oklahoma reared under identical conditions. The VLP were not transmissible to any of the seven plant hosts (in four families) of F. fusca suggesting that they may be an insect virus. Transmission of tomato spotted wilt virus (TSWV) by F. fusca, a known vector, was not affected by the presence or absence of the VLP. No TSWV particles were detected in tissues of F. fusca that transmitted TSWV to test plants. The VLP occurred in several internal organs and hemocoele of the thrips and were isolated in vitro by preparing homogenates of gut tissues. Infection of oocytes and presence of VLP in young nymphs suggested transovarial transmission of the particles. The VLP measured 62 ± 4 nm in diameter and usually occurred in dense viroplasms in the cell cytoplasm. Development of the particles within the viroplasms is discussed.     

Ultrastructural description of new Rickettsia-like organisms in the commercial abalone Haliotis tuberculata (Gastropoda: Haliotidae) from the NW of Spain

Rickettsia-like organisms (RLOs) were found in the commercially farmed abalone Haliotis tuberculata in the northwestern region of the Atlantic Coast of Spain and are described from light and transmission electron microscopy observations. The RLOs measured approximately 1.6 x 0.9 microm and were found in intracytoplasmic, spherical to ellipsoidal vacuoles (up to 8 microm) in the epithelial cells of the digestive diverticulae. The morphological ultrastructure of these organisms was typically prokaryotic, including a plasmalemma and a thin Gram-negative type cell wall. Several ultrastructural changes were observed in the epithelial cells of the host containing the RLOs. The nuclei became pycnotic and several basophilic dense inclusions appeared in the cytoplasm. In addition, the host cell appeared lysed and was ruptured in advanced stages of infection. It was impossible to ascertain whether the RLOs are responsible for this disease, as a haplosporidian infection was also present. We can only conclude that the presence of RLOs simultaneously with a haplosporidian parasite may contribute to the mortality of the abalone host.     

Prevalence of parasites in amphipods Diporeia spp. from Lakes Michigan and Huron, USA

Amphipods of Diporeia spp. have declined considerably during the last decade in the Great Lakes. We examined the possibility that disease may be affecting these populations. A histological survey assessed the parasites in species of Diporeia within Lakes Huron and Michigan, USA, and the host response to some of them and to unknown factors. Amphipods were found to have an intranuclear inclusion body, and were hosts to a rickettsia-like organism, fungi, a haplosporidian, a microsporidian, epibiotic ciliates, a gregarine, a cestode, acanthocephalans and nodule formations. Epibiotic ciliates were most common (37% prevalence of infection), but a microsporidian (3.8%), a rickettsia-like organism (1.6%), fungi, including a yeast-like organism (1.3%), worms (1.3%), and a haplosporidian (0.7%) are likely associated with mortalities or detrimental effects on the host. The role these agents may have played in the decline of Diporeia spp. in the Great Lakes over the last decade is not clear. Interrelationships with the dynamics of various physical and biological factors such as high sedimentation, diminished food supplies, and virulent parasites could synergistically cause the decline in Diporeia spp. populations in Lakes Michigan and Huron.     

Infectivity, transmission and 16S rRNA sequencing of a rickettsia, Coxiella cheraxi sp. nov., from the freshwater crayfish Cherax quadricarinatus

A rickettsia-like organism isolated from infected, farm-reared Cherax quadricarinatus was cultured in the yolk sac of developing chicken eggs, but could not be cultured in 3 continuous cell lines, bluegill fry (BF-2), fathead minnow (FHM), and Spodoptera frugiperda (Sf-9). The organism was confirmed by fulfilling Koch's postulates as the aetiological agent of mortalities amongst C. quadricarinatus. When C. quadricarinatus was inoculated with the organism, mortality was 100% at 28 degrees C and 80% at an ambient temperature of 24 degrees C. Horizontal transmission with food and via the waterborne route was demonstrated, but mortalities were lower at 30 and 10% respectively over a 4 wk period. The 16S rRNA sequence of 1325 base pairs of the Gram-negative, obligate intracellular organism was 95.6% homologous to Coxiella burnetii. Of 18 species compared to this rickettsia, the next most closely related bacterium was Legionella pneumophila at 86.7%. The suggested classification of this organism is Order Rickettsiales, family Rickettsiaceae, tribe Rickettsieae, within the genus Coxiella. We suggest it should be named Coxiella cheraxi sp. nov.     

Identification of expressed genes in cDNA library of hemocytes from the RLO-challenged oyster, Crassostrea ariakensis Gould with special functional implication of three complement-related fragments (CaC1q1, CaC1q2 and CaC3)

A SMARTer? cDNA library of hemocyte from Rickettsia-like organism (RLO) challenged oyster, Crassostrea ariakensis Gould was constructed. Random clones (400) were selected and single-pass sequenced, resulted in 200 unique sequences containing 96 known genes and 104 unknown genes. The 96 known genes were categorized into 11 groups based on their biological process. Furthermore, we identified and characterized three complement-related fragments (CaC1q1, CaC1q2 and CaC3). Tissue distribution analysis revealed that all of three fragments were ubiquitously expressed in all tissues studied including hemocyte, gills, mantle, digestive glands, gonads and adductor muscle, while the highest level was seen in the hemocyte. Temporal expression profile in the hemocyte monolayers reveled that the mRNA expression levels of three fragments presented huge increase after the RLO incubation at 3 h and 6 h in post-challenge, respectively. And the maximal expression levels at 3 h in post-challenge are about 256, 104 and 64 times higher than the values detected in the control of CaC1q1, CaC1q2 and CaC3, respectively.     

Multi response biomarker approach in the crab Carcinus aestuarii experimentally exposed to benzo a pyrene, polychlorobiphenyls and methyl mercury

The aim of this study was to test a multi response biomarker approach for evaluating toxicological risk due to some of the main contaminants in the Mediterranean benzo a pyrene, polychlorobiphenyls and methyl mercury , using the Mediterranean crab Carcinus aestuarii as bioindicator organism. Forty crabs were injected with different doses of these contaminants. Several molecular, biochemical and genotoxic biomarkers were tested in different tissues and biological materials. The main conclusions were: 1 hepatopancreas, gills, haemolymph and excreta seem to be useful for biomarker studies in this species; 2 several biochemical, molecular and genotoxic biomarkers were found suitable for testing in these tissues; 3 several biomarkers were found suitable for evaluating chemical stress due to different Mediterranean contaminants.     

Hermit crabs and their symbionts: Reactions to artificially induced anoxia on a sublittoral sediment bottom

Hermit crabs play an important role in the Northern Adriatic Sea due to their abundance, wide range of symbionts, and function in structuring the benthic community. Small-scale (0.25?m(2)) hypoxia and anoxia were experimentally generated on a sublittoral soft bottom in 24?m depth in the Gulf of Trieste. This approach successfully simulates the seasonal low dissolved oxygen (DO) events here and enabled studying the behaviour and mortality of the hermit crab Paguristes eremita. The crabs exhibited a sequence of predictable stress responses and ultimately mortality, which was correlated with five oxygen thresholds. Among the crustaceans, which are a sensitive group to oxygen depletion, P. eremita is relatively tolerant. Initially, at mild hypoxia (2.0 to 1.0?ml?l(-?1) DO), hermit crabs showed avoidance by moving onto better oxygenated, elevated substrata. This was accompanied by a series of responses including decreased locomotory activity, increased body movements and extension from the shell. During a moribund phase at severe hypoxia (0.5 to 0.01?ml?l(-?1) DO), crabs were mostly immobile in overturned shells and body movements decreased. Anoxia triggered emergence from the shell, with a brief locomotion spurt of shell-less crabs. The activity pattern of normally day-active crabs was altered during hypoxia and anoxia. Atypical interspecific interactions occurred: the crab Pisidia longimana increasingly aggregated on hermit crab shells, and a hermit crab used the emerged infaunal sea urchin Schizaster canaliferus as an elevated substrate. Response patterns varied somewhat according to shell size or symbiont type (the sponge Suberites domuncula). Mortality occurred after extended anoxia (~?1.5?d) and increased hydrogen sulphide levels (H(2)S ~?128?μmol). The relative tolerance of crabs and certain symbionts (e.g. the sea anemone Calliactis parasitica) - as potential survivors and recolonizers of affected areas - may influence and promote community recovery after oxygen crises.     

Restriction fragment length polymorphism differences among Illinois long-term selection oil strains

Restriction fragment length polymorphism (RFLP) analysis was used to characterize variability in the Illinois Long-Term Selection Experiment oil strains. Considerable polymorphism was detected within each oil strain and among oil strains. Fifty-two individual plants from each of the Illinois High Oil (IHO), Illinois Low Oil (ILO), Reverse High Oil (RHO) and Reverse Low Oil (RLO) strains were sampled to determine RFLP allele/variant frequencies. Generation 90 was sampled for IHO, RHO, and RLO whereas generation 87 was sampled for ILO. Forty-nine RFLP probes distributed throughout the maize genome were used. Chi-square analysis was performed to determine if RFLP genotypes at each of the 49 RFLP loci were significantly different among strains. Oil strains that have been separated for 90 generations showed high levels of significantly-different RFLP genotypic frequencies. The comparison of ILO vs RHO gave only significant chi-square values while the comparisons of IHO vs RLO and RHO vs RLO had 111 ratios of significant to non-significant chi-square values. Strains that have been separated for only 42 generations showed a lower level of significantly-different RFLP genotypic frequencies. The comparisons of IHO vs RHO and ILO vs RLO both had only a 32 ratio of significant to non-significant chi-squares values. Detection of multiple RFLP alleles/variants among the oil strains was common with 59% of the RFLP loci examined exhibiting multiple variants. A number of RFLP loci in RHO (3) and RLO (11) were associated with a trend in RFLP allele/variant frequencies consistent with a response to reverse selection for oil concentration.