The acceleration of linear DNA during pulsed-field gel electrophoresis

The velocity and orientation of T4 and lambda DNA have been measured for the first 20 s during pulsed-field gel electrophoresis in order to clarify the DNA motions that occur. For a square pulse with field strength E = 10 V/cm, the velocity of lambda DNA increases gradually to 10.5 microns/s in 1.0 s, declines to 8.6 microns/s, and then rises to a plateau value of 9.3 microns/s after 4 s. T4 DNA behaves similarly, but more slowly. Parallel measurements of fluorescence-detected linear dichroism show that the DNA becomes substantially aligned with its chain axis parallel to the electrophoretic field E after the pulse is applied. The alignment also shows an overshoot, an undershoot, and a plateau comparable to those seen for velocity. When the field strength increases, both the velocity and the alignment reach their peaks more quickly. For all field strengths and both molecular weights, the velocity peak occurs when the molecular center of mass has moved 0.3 to 0.5 L, where L is the chain contour length. A qualitative model is provided.     

Characterization and taxonomic status of tick spiroplasmas: a review

Three serologically distinct groups of spiroplasmas have been recovered from ticks. Spiroplasma mirum strains (from rabbit ticks, Haemaphysalis leporispalustris) and Y32 group (VI) spiroplasmas (from Ixodes pacificus) are the only spiroplasmas to have a clear association with these arthropods. Group (VI) spiroplasmas are distinguished by an unusual nonhelical morphology and their capacity to hemadsorb guinea pig erythrocytes. S. mirum strains are unique in their ability to induce cataracts or lethal brain infections in a number of young vertebrates and in their virulence for the chick embryo. The 277F spiroplasma, while initially recovered from a pool of rabbit ticks (H. leporispalustris), is related by certain serological and genetic properties to spiroplasmas in the S. citri complex (serogroup I). These relationships suggest that the 277F spiroplasma may not be a natural inhabitant of the rabbit tick.     

Improved Cultivation Systems for Isolation of the Colorado Potato Beetle Spiroplasma

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.     

Direct isolation in cell-free medium of a spiroplasma fromHaemaphysalis leporispalustris (Acari: Ixodidae) in Maryland

A spiroplasma isolate, was obtained from rabbit ticks (Haemaphysalis leporispalustris) taken from cottontail rabbits in Maryland by inoculation of tick suspensions into SP-4 medium. The isolate was indistinguishable from an experimental vertebrate pathogen (suckling mouse cataract agent spiroplasma) when tested with other plant and tick spiroplasmas in growth inhibition, deformation, and metabolism inhibition tests. The isolated organism had a pathogenic profile for suckling rats and embryonated chicken eggs that differed significantly from that of other suckling mouse cataract agent strains. This is the first report of a direct spiroplasma isolation from ticks in cell-free medium, and confirms the specific association of spiroplasmas of the suckling mouse cataract agent serogroup with rabbit ticks.     

Lampyridae (Coleoptera): A plethora of mollicute associations

Beetles (Coleoptera) harbor many species ofAcholeplasma andSpiroplasma (division Tenericutes, class Mollicutes). Mollicutes were isolated from guts and/or hemocoels of firefly beetles (Lampyridae) from the United States (Maryland and West Virginia), Ecuador, and Tobago. Firefly beetles were frequent hosts for the group XIV spiroplasma, isolated from Ellychnia corrusca, and the group XIX spiroplasma, isolated fromPhoturis spp. The most unusual feature of the firefly-mollicute association is the carriage of four Mycoplasma species. Recent phylogenetic studies indicate that these species are members of a clade that includes a vertebrate pathogen,Mycoplasma mycoides. The high rate of occurrence ofMycoplasma species (which are, otherwise, infrequent in insects) in lampyrid beetles suggests that the association is significant. The unusual light-producing physiology of lampyrids (which is dependent on large pools of energy) and the production of large amounts of cardenolides from cholesterol (a critical growth factor for many mollicutes) may favor colonization by mollicutes. Offprint requests to: K. J. Hackett.     

Wall-less prokaryotes from fall flowers in central United States and Maryland

Four spiroplasma strains and eleven isolates tentatively identified as acholeplasmas were obtained from fall flowers in Colorado, Nebraska, Illinois, and Maryland. Although the acholeplasma isolates were heterogeneous, all showed antigenic sharing with a group of unnamed organisms (L1 and related strains) isolated in othe studies from flowers in Florida. The W20 and W24 isolates from Nebraska were partially related to the L1 group by DNA-DNA homology and polyacrylamide gel electrophoresis (PAGE) analyses. A Colorado spiroplasma (W13) was identifed as a new strain of group IV complex. Three spiroplasma strains from flowers in Maryland old fields represent a new serovar with closest affinity to subgroup I-4 and to the LB12 and N525 serovars of group I. Widespread occurrence of acholeplasmas on flowers in this study, and on plant surfaces in general, suggests that, like spiroplasmas they probably will be found to reside in arthropods.     

Spiroplasma species in the Costa Rican highlands: implications for biogeography and biodiversity

More than 1,000 Spiroplasma isolates have been obtained from horse flies and deer flies (Diptera:Tabanidae) in the United States and Canada. However, the spiroplasma biota of Central America is poorly known. In August of 1995 and 1998, 13 isolates were obtained in 14 attempts from horse flies of a single species, Poeciloderas quadripunctatus, taken in the Costa Rican highlands (1,100–2,000 m). The majority of the “isolates” proved to be mixtures of two or more Spiroplasma species, but after filter cloning, single strains emerged that were designated as representatives of the 13 accessions. Six distinct spiroplasma serogroups were identified from these isolations. Three of the strains are putative new species with no serological relationship to any other Spiroplasma species. A fourth strain is a putative new species that may be distantly related to S. helicoides, a southeastern U.S. species. These four strains are accorded herein status as representatives of new serogroups: strain BARC 4886 (group XXXV); strain BARC 4900 (group XXXVI); strain BARC 4908 (group XXXVII); and GSU5450 (group XXXVIII). A fifth Spiroplasma species was very closely related to S. lineolae, known previously only from the Georgia (U.S.) coast. The sixth was most closely related to subgroup VIII-3, known from Texas and the southeastern U.S. Discovery of six spiroplasma species in only 13 attempted isolations reflects diversity seldom equaled in southeast Georgia, and never elsewhere in the U.S. These results are consistent with a hypothesis that spiroplasma diversity increases from north (Nova Scotia) to south (Georgia and Costa Rica). The discovery of significant affinity between some spiroplasmas of the southeastern U.S. and the Costa Rican highlands was unexpected, but may reflect a climatically complex Pleistocene history.

Assessing diversity of arbuscular mycorrhizal fungi in a local community: role of sampling effort and spatial heterogeneity

Diversity of arbuscular mycorrhizal fungi (AMF) was assessed in two 9.2 × 9.2-m plots planted with landscape trees and shrubs at an experimental site in Phoenix, AZ, USA. Twenty-five soil samples were collected in a regular grid pattern from each plot, and AMF species were identified using trap cultures. A total of 12 species were detected, with 7 species detected in one plot and 11 in the other. We found that sampling effort had a major impact on assessing species richness and composition in this local community. Fifteen samples would be necessary to detect 70–80% of species present in each plot. A limited number of additional undetected species are likely to be present in both plots, based on the sampling effort curves and jackknife estimates. Only two species, Glomus eburneum and Glomus microaggregatum, were detected in over 50% of the samples from both plots, and rank–frequency plots revealed a lognormal species distribution. Despite the patchiness of plants in the plots, the number of species detected per point exhibited spatial structuring only at the smallest sampling scale in a single plot, and only a single species in each plot was not randomly distributed. These results indicate that sampling effort and strategy can affect perceptions of AMF community structure.     

Influence of immune‐relevant genotype on the reproductive success of a salmonid alternative mating strategy

Major histocompatibility complex (MHC) and immune‐relevant gene markers were used to evaluate differences in reproductive success (RS) among naturally spawning coho salmon Oncorhynchus kisutch mate pairs involving an alternative male reproductive phenotype, known as jacks. These mate pairs included both hatchery‐reared and wild origin fish such that three classes were evaluated in two consecutive years (2005 and 2006) using a previously constructed multigenerational genetic pedigree: wild × wild (W × W), hatchery × hatchery (H × H) and wild × hatchery (W × H). Oncorhynchus kisutch jack mate pairs mated randomly based on immune‐relevant genotype in both years; a result consistent with the opportunistic mating strategy of jacks. An association between greater number of alleles shared at three immune‐relevant gene markers and increased RS was found for: W × H mate pairs in 2005 (BHMS429), W × H pairs in 2006 (SsalR016TKU) and W × W pairs in 2006 (OMM3085). No correlation between immune gene diversity and RS was found for H × H pairs in either year. The results suggest that the influence of immune‐relevant genotype on mating success may be different for jacks when compared with previous studies of large adult male O. kisutch.