Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

Genetic diversity and population structure of spottedtail goby (Synechogobius ommaturus) based on AFLP analysis

Larval dispersal may have an important impact on genetic structure of benthic fishes. To examine population genetic structure of spottedtail goby Synechogobius ommaturus, samples from five different locations of China and Kunsan population in Korea were analyzed by using amplified fragment length polymorphism (AFLP) technology. A total of 253 bands were identified from 91 individuals by 5 primer combinations and the percentage of polymorphic bands was 43.87%. The average gene diversity was 0.0794 ± 0.1470 and Shannon’s information index was 0.1279 ± 0.2138. The pairwise F_st values ranged from 0.022 to 0.201. The results of AMOVA analysis indicated that 90.54% of the genetic variation contained within populations and 9.46% occurred among populations. The gene flow estimates (Nm) demonstrated that different gene flow existed among populations from 0.994 to 11.114. No significant genealogical branches or clusters were recognized on the UPGMA tree. The results support the hypothesis that larval dispersal ability can be responsible for the genetic diversity and population structuring.     

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group (r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.     

The genetic analysis of F1 hybrid larvae between female Trichinella spiralis and male Trichinella britovi

Hybrids between female Trichinella spiralis and male Trichinella britovi were constructed. Then, hybrid genotype was characterized by DNA markers including mitochondrial cytochrome c oxidase subunit I (CO I) gene, the gene encoding the 43-kDa excretory–secretory (ES) protein, and genomic DNA fragments specific for T. spiralis and T. britovi identified from random amplified polymorphism DNA (RAPD). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial CO I gene revealed that all hybrids carried a T. spiralis pattern. The same analysis of the gene encoding the 43-kDa ES protein showed that each hybrid carried both T. spiralis and T. britovi gene type simultaneously. In the analysis of genomic DNA using RAPD-derived PCR primers, some hybrids carried T. spiralis and T. britovi-specific RAPD markers, while others carried the RAPD marker of T. spiralis only.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

An AFLP analysis of genetic diversity and structure of Caragana microphylla populations in Inner Mongolia steppe, China

Genetic diversity and structure of five natural populations of Caragana microphylla from the Inner Mongolia steppe were estimated using AFLP markers. Five pairs of primers generated a total of 312 bands among 90 individuals, with percentage of polymorphic bands (PPB) being 63% at the population level and 76% at the species level, respectively. The genetic diversity within populations was correlated significantly with the soil N:P ratio. AMOVA analysis revealed high genetic variations within populations (95.5%). The estimated number of migrants per generation (Nm) was 10.72, indicating a high level of gene flow among populations. There was no significant correlation (r = 0.36) between genetic distance and geographical distance. These results were discussed in terms of eco-geographical variations among populations, together with the life history traits and breeding system of the species. The knowledge obtained may have important implications for better conservation and wise use of the vegetation dominate by C. microphylla.     

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.     

Host preference in Pseudacteon phorid flies: response of P. tricuspis and P. curvatus to black, red and hybrid imported Solenopsis fire ants in multiple choice bioassays

Host preferences in both sexes of Pseudacteon tricuspis Borgmeier (Jaguariuna biotype) and Pseudacteon curvatus Borgmeier (Formosa biotype) and their relative attraction to the imported fire ants (IFA), Solenopsis invicta Buren (red IFA), Solenopsis richteri Forel (black IFA) and S. invicta × S. richteri hybrids (hybrid IFA) were investigated in two separate experiments utilizing multiple choice flight bioassays. The results of both experiments clearly showed that both sexes of the Jaguariuna biotype of P. tricuspis could distinguish among the three IFA species and demonstrated greater preference for hybrid IFA and red IFA. This conclusion is supported by a variety of data collected on the number of fly visits, attack rate, and hovering duration (Experiment 1), and on the number of trapped flies (Experiment 2), which showed that black IFA is the least preferred of the three species. Similar results were recorded for the Formosan biotype of P. curvatus, although the data were not as strongly conclusive. Females of this biotype spent a significantly greater amount of time in hovering mode over red IFA and hybrid IFA compared to black IFA, but the other data were not significant. The red IFA is the natural host of both phorid fly biotypes and our results suggest that both biotypes may have evolved a specialized relationship with red IFA including an ability to discriminate it from related fire ants. These results are discussed in relation to the possible role of fire ant chemicals in mediating host preferences in phorid flies, contributions of male phorid flies to fire ant biocontrol, and the practical implications of the key findings.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae)

Tetradenia riparia (Lamiaceae) is a well-known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of T. riparia essential oil (EO) against engorged females of Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). For this purpose, nine serial concentrations (12.50%, 6.25%, 3.75%, 1.80%, 0.90%, 0.45%, 0.22%, 0.11%, and 0.056% w/v) of T. riparia were used for the adult immersion test (AIT). For the larval packet test (LPT), we used 14 serial concentrations (100.00%, 50.00%, 25.00%, 12.50%, 6.25%, 3.65%, 1.82%, 0.91%, 0.45%, 0.228%, 0.114%, 0.057%, 0.028%, and 0.014% w/v). The results for AIT showed 100.00% and 2.05% mortality, 19.00 and 90.20% for the total number of eggs, egg-laying inhibition of 0.00% and 90.20%, hatchability inhibition of 0.00% and 70.23%, and product effectiveness of 100.00% and 2.89%, respectively. The AIT indicated that the LC₅₀ and LC_99.9, calculated using the Probit test, were for mortality (%) 0.534 g/mL (0.436–0.632) and 1.552 g/mL (1.183–1.92); for total number of eggs were 0.449 g/mL (0.339–0.558) and 1.76 g/mL (1.27–2.248); and for hatchability inhibition were 0.114 g/mL (0.0–0.31) and 2.462 g/mL (1.501–3.422), respectively. Larvae between 14 and 21 days old were fasted and placed in each envelope. Bioassays were performed at 27° ± 1 °C, RH ? 80%. Larval mortality was observed 24 h after treatment and showed 10.60–100% mortality in the LPT bioassay. The LPT showed that the LC₅₀ and LC_99.9 were 1.222 g/mL (0.655–1.788) and 11.382 g/mL (7.84–14.91), respectively. A positive correlation between T. riparia EO concentration and tick control, was observed by the strong acaricidal effects against R. (B.) microplus, and the mortality rate of ticks was dose-dependent. Our results showed that T. riparia is a promising candidate as an acaricide against resistant strains of R. (B.) microplus.     

Genetic relationships among Mexican white pines (Pinus, Pinaceae) based on RAPD markers

Genetic relationships among Mexican white pines have not been completely resolved by DNA sequencing analyses. The use of random amplified polymorphic DNA (RAPD) markers for the study of interspecific relationships has been questioned because of the possible lack of homology of co-migrating bands between species. However, several RAPD based studies on pines have provided sufficient information to discriminate between closely related taxa. Genetic relationships among four species of Mexican white pines (Pinus ayacahuite, Pinus strobiformis, Pinus lambertiana and Pinus chiapensis) were estimated based on RAPD markers. Sixty-nine primers generated 247 bands in pooled DNA samples from ten populations. In addition, four selected primers generated 27 bands in 176 individual DNA samples. Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendrograms based on Jaccard similarity indices were constructed. The results suggest that the closest pine species analyzed were P. ayacahuite and P. strobiformis, followed by P. lambertiana. The most genetically distant species was P. chiapensis. Cluster analyses did not support P. strobiformis as a distinct species from P. ayacahuite.     

Population genetic structure of striped mullet, Mugil cephalus, along the coast of China, inferred by AFLP fingerprinting

The striped mullet, Mugil cephalus, is an economically important species for both aquaculture and commercial fisheries in China. In this study, the amplified fragment length polymorphism (AFLP) technique was employed to analyze population genetic diversity and genetic distance between different populations with the aim of elucidating the population structure and gene flow of M. cephalus along the coast of China. A total of 230 fragments with 150–500 bp were identified from 118 individuals by five AFLP primer combinations. The polymorphic loci within populations varied from 46.52% to 64.78%, with an average of 53.91%, and the average heterozygosity from 0.1829 to 0.2282, with an average of 0.2074. The UPGMA phenograms of 118 individuals were constructed based on Dice similarity coefficients and four clusters were recognized. AMOVA analysis revealed that 60.7% of genetic variations were within populations and 39.3% between populations. The estimated genetic distance (φ_ST) value over all polymorphic loci across the six populations was 0.393 (p < 0.0010), indicating a strong population structuring. The pairwise φ_ST value ranged from 0.1112 to 0.5358, with an average of 0.3693. The population pairwise gene flows (average N_m = 0.73) are low. In addition, the result of the Mantel test showed that there was a significant correlation between geographic and genetic distances (r = 0.5434, p = 0.0050). It was speculated that there exist at least four distinct geographic populational subdivisions of M. cephalus along the Chinese coast. This research has provided new molecular data which will aid our understanding of the genetic structure of this species.     

Molecular phylogeny of the Acre clade (Crassulaceae): Dealing with the lack of definitions for Echeveria and Sedum

The phylogenetic relationships within many clades of the Crassulaceae are still uncertain, therefore in this study attention was focused on the “Acre clade”, a group comprised of approximately 526 species in eight genera that include many Asian and Mediterranean species of Sedum and the majority of the American genera (Echeveria, Graptopetalum, Lenophyllum, Pachyphytum, Villadia, and Thompsonella). Parsimony and Bayesian analyses were conducted with 133 species based on nuclear (ETS, ITS) and chloroplast DNA regions (rpS16, matK). Our analyses retrieved four major clades within the Acre clade. Two of these were in a grade and corresponded to Asian species of Sedum, the rest corresponded to a European–Macaronesian group and to an American group. The American group included all taxa that were formerly placed in the Echeverioideae and the majority of the American Sedoideae. Our analyses support the monophyly of three genera – Lenophyllum, Thompsonella, and Pachyphytum; however, the relationships among Echeveria, Sedum and the various segregates of Sedum are largely unresolved. Our analyses represents the first broad phylogenetic framework for Acre clade, but further studies are necessary on the groups poorly represented here, such as the European and Asian species of Sedum and the Central and South American species of Echeveria.     

Genetic modification in the cyclin gene from a Nicotiana interspecific hybrid: role of the GTcyc gene in tumorization process

The organistic constitution of genetic tumors probably causes the constituent cells to undergo genetic change from normal growth to abnormal, a relatively undifferentiated proliferation. We report here that the cyclin GTcyc gene, isolated from genetic tumors yielded notably intense bands while those from the parental DNA were less expressed. In a similar fashion, Northern blot analysis revealed that the genetic tumors expressed high levels of GTcyc relative to non-tumor hybrid tissues. Furthermore, RAPD data showed that the genetic relationships between tumor tissues and their parents did not present a highly corresponding match, suggesting that tumor growth may relate to the genetic modification or hybridization-related genome reorganization. Taken together, the cyclin gene performs a critical role in cell cycle progression, and this particular gene (GTcyc) may be a potential factor in tumor formations, resulting in gene alterations or gains, or changes to specific genomic regions.     

Biological control of brown rot (Moniliana laxa Ehr.) on apricot (Prunus armeniaca L. cv. Hacıhaliloğlu) by Bacillus, Burkholdria, and Pseudomonas application under in vitro and in vivo conditions

In 2002 and 2003, a study was conducted to determine the effect of bacterial strains, Burkholdria OSU 7, Bacillus OSU 142, and Pseudomonas BA 8, on biological control of brown rot disease (Monilinia laxa Ehr.) on apricot cv. Hacıhaliloğlu in Malatya province of Turkey. Apricot orchard at full blooming stage was inoculated with conidial suspension (1 × 10⁶ spores/ml) of M. laxa Ehr. After inoculation, two apricot trees for each application were treated with each of the three biological control agents (Burkholdria gladii OSU 7, Bacillus subtilis OSU 142, and Pseudomonas putida BA 8) by spraying (1 × 10⁹ cfu/ml) on inoculated branches. Disease incidence was evaluated for untreated (control 1) and four different treatment groups including commercial disease management (control 2, positive control: 3% Bourdox in fall, 50% Cupper at pink flower, 30 g/100 l Corus at first blooming, and 300 g/100 l Captan at last blooming stage) and treatments including each of the three bacterial strains (OSU 7, OSU 142, and BA 8). The results showed that disease incidence for negative control (control 1) was 9.94, which was significantly higher than disease incidence for commercial application (2.57%) or bacterial treatments (2.82–5.00%) in the first year. In 2003, the lowest disease incidence observed in OSU 7 treatment (6.80%), while disease incidence rate for positive control and negative control were 9.45% and 28.46%, respectively. This result may suggest that OSU 7 has potential to be used as biopesticide for effective management of brown rot disease on apricot.     

A molecular and morphological investigation of Batrachospermum arcuatum (Batrachospermales, Rhodophyta) in China

Batrachospermum arcuatum specimens were analysed from seven stream segments in North China. Morphological characteristics were observed and cluster analysis was used to evaluate the divergence among thalli from. Sequence data of the rbcL gene (chloroplast gene) and cox2-3 spacer region (mitochondrial gene) were also utilized to evaluate genetic variation in specimens among stream segments. The specimens from four of the streams were monoecious, while the individuals at the other three locations were dioecious. Cluster analysis showed that the monoecious specimens were not separated from the dioecious specimens, based on morphology, but rather the specimens were grouped by geographical closeness and habitat similarity. Likewise, the combined analyses of rbcL and the cox2-3 spacer data from provided more evidence that breeding system (monoecy vs. dioecy) is not a good morphological character to distinguish species.     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Assessment of the genetic diversity among Glyptosternum maculatum, an endemic fish of Yarlung Zangbo River, Tibet, China using SSR markers

The genetic diversity of Glyptosternum maculatum populations from Nyang River, Lhasa River, and Shetongmon Reach of Yarlung Zangbo River was assessed using six microsatellite markers. Overall, the genetic diversity across the three populations was low. The Shetongmon population exhibited the highest level of genetic diversity in terms of number of alleles and effective alleles, heterozygosity, and polymorphic information content value, followed by the Nyang population and Lhasa population. The analysis of molecular variance demonstrated that almost the variation (86.64%) occurred within populations. The differentiation among populations was not significant, and population structure was weak. These results revealed that three natural populations of G. maculatum are not genetically differentiated and the large disparity of living altitude did not caused genetic differentiation between different populations. Our observations will help identify the genetic relationship among populations to understand the genetic diversity of G. maculatum.