Genetic diversity, population structure and relationships of Tunisian Thymus algeriensis Boiss. et Reut. and Thymus capitatus Hoffm. et Link. assessed by isozymes

The genetic diversity and population structure of two Tunisian Thymus species (Thymus algeriensis and Thymus capitatus), from 46 natural populations growing in six bioclimates, were analysed by starch gel electrophoresis using eight isozymes. The genetic diversity within populations varied at species level. Variation in T. algeriensis was higher than that observed for T. capitatus, and exclusive alleles were detected for the two studied species. A high differentiation among populations, for each species, estimated by Wright's F-statistics was revealed. In each species, a high level of inbreeding within populations induced by Wahlund effect was observed. A relatively high level of differentiation associated with a restricted gene flow among species was revealed. The PCA and UPGMA analysis, performed on all populations, showed two distinct groups with respect of specific differentiation level. The high genetic divergence between the two species corroborates their taxonomic status, as previously reported using morphological traits. The strategy for the management and conservation of populations should be made for each taxa according to its level of diversity and bioclimate.     

Characterization of 12 Novel Microsatellite Markers of Sogatella furcifera (Hemiptera: Delphacidae) Identified From Next-Generation Sequence Data

The white-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae), is a major pest of rice and has long-range migratory behavior in Asia. Microsatellite markers (simple sequence repeats) have been widely used to determine the origins and genetic diversity of insect pests. We identified novel microsatellite loci for S. furcifera samples collected from Laos, Vietnam, and three localities in Bangladesh from next-generation Roche 454 pyrosequencing data. Size polymorphism at 12 microsatellite loci was verified for 40 adult individuals collected from Shinan, South Korea. The average number of alleles per locus was 7.92. The mean values of observed (H_o) and expected heterozygosities (H_E) were 0.615 and 0.757, respectively. These new microsatellite markers will be a resource for future ecological genetic studies of S. furcifera samples across more broad geographic regions in Asia and may assist in estimations of genetic differentiation and gene flow among populations for implementation of more effective management strategies to control this serious rice pest.     

Identification of the Population Structure of Myzus persicae (Hemiptera: Aphididae) on Peach Trees in China Using Microsatellites

In this study, we characterized the genetic structure of Myzus persicae (Sulzer) (Hemiptera: Aphididae) populations in China using microsatellites. We expected that these data will reveal the genetic relationships among various populations of M. persicae and will be of value in the development of better methods for pest control. Four hundred sixty individuals from 23 areas over 13 provinces were collected in the early spring of 2010, all from their primary host, Prunus persicae. The markers analyzed were highly polymorphic, as demonstrated by the expected heterozygosity value (H_e = 0.861) and the Polymorphism Information Content (PIC = 0.847), which indicated that M. persicae maintains a high level of genetic diversity. Analysis of molecular variance revealed an intermediate level of population differentiation among M. persicae populations (F_ST = 0.1215). Geographic isolation existed among these populations, and, consequently, the genetic structure of the populations was split into a southern group and a northern group divided by the Yangtse River.     

Chloroplast DNA molecular characterization and leaf volatiles analysis of mint (Mentha; Lamiaceae) populations in China

Non-coding chloroplast DNA (cpDNA) was used to evaluate genetic diversity and relationships within the section Mentha collected from China. Leaf volatiles of Mentha spicata accessions and its parent were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). To this end, genetic diversity of 30 Mentha accessions from different geographic origins of China, representing six species and two hybrids, was assessed. Molecular studies grouped the samples mainly according to genetic relationship established by conventional methods. Cluster analysis based on the leaf volatiles chemical composition of M. spicata accessions defined two main chemotypes: 1,8-cineole-piperitenone oxide type and limonene-carvone type. The results suggested that there was a high genetic variability among individuals of Mentha in China. A distinct correlation between cpDNA marker and volatile oils in M. spicata accessions was revealed. Moreover, the chemical composition followed maternal inheritance in M. spicata accessions. In conclusion, the genetic diversity of Mentha populations as shown in this study should play a critical role in future selection and breeding programs.     

Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41

Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg²⁺ binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.     

A comparison of monitoring systems used for Ceratitis species (Diptera: Tephritidae) in South Africa

For 16 years a bucket-type trap known as the Sensus fruit fly trap has been used to monitor three fruit fly pest species Ceratitis capitata, Ceratitis rosa and Ceratitis cosyra in South African fruit industries. The relative efficiencies of lures sold for monitoring fruit flies with the Sensus trap in South Africa were determined in field experiments where laboratory-reared C. capitata, C. rosa and C. cosyra were released in a mango orchard within a few metres of Sensus traps containing either Capilure (trimedlure or tert-butyl 4 (or 5) -chloro-2-methylcyclohexane carboxylate), Ceratitislure (protein hydrolysate plus β-caryophyllene) or Questlure (protein hydrolysate plus plant extracts). When using 12-day-old flies, Capilure caught 3 times more C. capitata males than C. rosa males and this difference was more extreme when 3-day-old flies were released. Ceratitislure caught significantly more 12-day-old C. cosyra males than 12-day-old C. capitata males, but the difference was reversed when 3-day-old flies were compared. Questlure showed the least differences between species and age but recovered the lowest proportion of released species. Further comparisons were conducted in an orchard with wild flies using other known attractants in larger yellow Probodelt bucket traps. Capilure caught more male C. capitata than BioLure Fruit Fly 3-component, but BioLure 2-component (trimethylamine and ammonium acetate) was more effective than Questlure for C. capitata females. The 3-component lure was also more effective than both Capilure and Questlure for male and female C. rosa, respectively. Ceratitislure was the most effective lure for male C. cosyra flies and BioLure 3-component was more effective than Ceratitislure and Questlure for female C. cosyra flies. The intervention threshold of 4 flies/trap/week previously used in citrus with Capilure for C. rosa was lowered to 2 C. rosa/trap/week when using the Sensus trap due to the lower sensitivity of this trap-lure combination found for C. rosa in this study. The 3-component lure, or the 2-component combination of trimethylamine and ammonium acetate in a ratio of 1:8 in the Sensus trap capsule, would be more effective for both sexes of all three Ceratitis species than the Questlure that is currently being used.     

Microsatellite data suggest significant population structure and differentiation within the malaria vector Anopheles darlingi in Central and South America

Background

Anopheles darlingi is the most important malaria vector in the Neotropics. An understanding of A. darlingi's population structure and contemporary gene flow patterns is necessary if vector populations are to be successfully controlled. We assessed population genetic structure and levels of differentiation based on 1,376 samples from 31 localities throughout the Peruvian and Brazilian Amazon and Central America using 5–8 microsatellite loci.

Results

We found high levels of polymorphism for all of the Amazonian populations (mean R _S = 7.62, mean H _O = 0.742), and low levels for the Belize and Guatemalan populations (mean R _S = 4.3, mean H _O = 0.457). The Bayesian clustering analysis revealed five population clusters: northeastern Amazonian Brazil, southeastern and central Amazonian Brazil, western and central Amazonian Brazil, Peruvian Amazon, and the Central American populations. Within Central America there was low non-significant differentiation, except for between the populations separated by the Maya Mountains. Within Amazonia there was a moderate level of significant differentiation attributed to isolation by distance. Within Peru there was no significant population structure and low differentiation, and some evidence of a population expansion. The pairwise estimates of genetic differentiation between Central America and Amazonian populations were all very high and highly significant (F _ST = 0.1859 – 0.3901, P < 0.05). Both the D _A and F _ST distance-based trees illustrated the main division to be between Central America and Amazonia.

Conclusion

We detected a large amount of population structure in Amazonia, with three population clusters within Brazil and one including the Peru populations. The considerable differences in N _e among the populations may have contributed to the observed genetic differentiation. All of the data suggest that the primary division within A. darlingi corresponds to two white gene genotypes between Amazonia (genotype 1) and Central America, parts of Colombia and Venezuela (genotype 2), and are in agreement with previously published mitochondrial COI gene sequences interpreted as incipient species. Overall, it appears that two main factors have contributed to the genetic differentiation between the population clusters: physical distance between the populations and the differences in effective population sizes among the subpopulations.     

Metabolomic Profiling and Genomic Study of a Marine Sponge-Associated Streptomyces sp

Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8) isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3) that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.     

Identification of Core Alpha 1,3-Fucosyltransferase Gene From Silkworm: An Insect Popularly Used to Express Mammalian Proteins

Silkworm has great potential as production system of recombinant mammalian proteins. When the protein products are used for medical purpose, it is required to reduce the risk of an allergy, the content of core alpha 1,3-fucosyl residue attached to the N-glycan of proteins, for example. We isolated the gene of an enzyme responsible for the transfer of core alpha 1,3-fucosyl residue, core alpha 1,3-fucosyltransferase (Fuc-T C3), from silkworm. A candidate cDNA for silkworm Fuc-T C3 was isolated as a homolog of the fruit fly enzyme gene fucTA. The gene was located on chromosome 7 of the silkworm genome and was composed of seven exons, which spanned approximately 10 kb on the genome. The coding region of the gene was 1,350 bp and encoded a 450-amino acid protein with a molecular mass of 52.2 kDa. Deduced amino acid sequence of the coding region showed one transmembrane domain in its N-terminal and typical motifs common to fucosyltransferases including Fuc-T C3s of other organisms in its C-terminal. The extract of CHO cells transfected with the cDNA showed Fuc-T C3 activity using GDP-fucose and DABS-GnGn peptide as substrates. These results showed this cDNA clone actually encodes silkworm Fuc-T C3.     

Influences of Cry1Ac Broccoli on Larval Survival and Oviposition of Diamondback Moth

Larval survival and oviposition behavior of three genotypes of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), (homozygous Cry1Ac-susceptibile, Cry1Ac-resistant, and their F₁ hybrids), on transgenic Bacillus thuringiensis (Bt) broccoli expressing different levels of Cry1Ac protein were evaluated in laboratory. These Bt broccoli lines were designated as relative low, medium, and high, respectively, according to the Cry1Ac content. Untransformed brocccoli plants were used as control. Larval survival of diamondback moth on non-Bt leaves was not significantly different among the three genotypes. The Cry1Ac-resistant larvae could survive on the low level of Bt broccoli plants, while Cry1Ac-susceptible and F₁ larvae could not survive on them. The three genotypes of P. xylostella larvae could not survive on medium and high levels of Bt broccoli. In oviposition choice tests, there was no significant difference in the number of eggs laid by the three P. xylostella genotypes among different Bt broccoli plants. The development of Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella on intact Bt plants was also tested in greenhouse. All susceptible P. xylostella larvae died on all Bt plants, while resistant larvae could survive on broccoli, which expresses low Cry1Ac protein under greenhouse conditions. The results of the greenhouse trials were similar to that of laboratory tests. This study indicated that high dose of Bt toxins in broccoli cultivars or germplasm lines is required for effective resistance management.     

Within- and Between-Species Variation of Wing Venation in Genus Monochamus (Coleoptera: Cerambycidae)

We have described the morphological variation of five Western Palaearctic species of Monochamus Dejean, 1821. The variation was assessed using wing measurements. Special emphasis was placed on the differences between Monochamus sartor (F., 1787) and Monochamus urussovii (Fischer-Waldheim, 1805). There was an interesting pattern of variation between the two species. Individuals of M. sartor from the Carpathians differed markedly from individuals of M. urussovii from Siberia, but individuals from north-eastern Poland were intermediate between those two populations. The intermediate individuals were more similar to the Siberian M. urussovii than to the Carpathian M. sartor despite the relatively large geographic distance between north-eastern Poland and Siberia. The occurrence of the intermediate individuals in north-eastern Poland may be the effect of hybridization between M. urussovii and M. sartor, which might have occurred after secondary contact between the two species in the Holocene.     

Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent

The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.     

Pre-harvest survival of codling moth in artificially infested sweet cherry fruits

Prior to the 2009 season, sweet cherry fruits, Prunus avium (L.) L., from North America were required to be fumigated with methyl bromide before being exported to Japan to eliminate possible infestation by codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). However, based on recent biological research on host status, a new procedure (the “systems approach”) was implemented relying on the cumulative effects of pre- and post-harvest practices to produce pest-free sweet cherry fruits for export. This is an organized process that involves the integration of procedures used in the production, harvest, packaging, and distribution of a commodity which cumulatively meet the requirements for quarantine security. The objective of our study was to provide additional information to support the systems approach for codling moth in sweet cherries fruits. For four seasons, branches with sweet cherry fruits were caged on trees and infested by released ovipositing codling moths into the cages. Fruits were sampled weekly for codling moth individuals at each life stage. Eggs were laid mostly on leaves with the seasonal average numbers per cage ranging from 142 to 617. Populations declined rapidly after egg eclosion, with <1.5% of the original cohort surviving to fifth instars. Even after 8 weeks, none had formed cocoons or pupated. This failure of the codling moth to complete a life cycle under field conditions supports previous life history studies and demonstrates that sweet cherry fruits are inadequate hosts for codling moth.     

Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.     

Efficacy of Plectranthus glandulosus (Lamiaceae) and Callistemon rigidus (Myrtaceae) Leaf Extract Fractions to Callosobruchus maculatus (Coleoptera: Bruchidae)

As part of on-going efforts to use eco-friendly alternatives to chemical pesticides, methanol crude extracts of Plectranthus glandulosus and Callistemon rigidus leaves were sequentially fractionated in hexane, chloroform, ethyl acetate, and methanol to establish the most active fraction(s) against Callosobruchus maculatus in cowpea. Cowpea seeds (25 g) were treated with 0.5, 1, 2, and 4 g/kg of extract to evaluate the contact toxicity and F₁ progeny production of the beetles in the laboratory. Mortality was recorded 1, 3, and 7 d postexposure. P. glandulosus hexane fraction was more toxic than the other fractions recording 100% mortality at 4 g/kg, within 7 d with LC₅₀ of 0.39 g/kg. Hexane fraction of C. rigidus showed superior toxicity, causing 100% mortality at 4 g/kg within only 1 d of exposure with LC₅₀ of 1.02 g/kg. All the fractions greatly reduced progeny emergence, with C. rigidus hexane fraction being the best progeny inhibitor. Fractions of P. glandulosus and C. rigidus leaves had sufficient efficacy to be a component of storage pest management package for C. maculatus.     

Conservation and storage of Curcuma amada Roxb. synseeds on Luffa sponge matrix and RAPD analysis of the converted plantlets

Curcuma amada Roxb. (family Zingiberaceae), is gaining global attention as potential source of new drug molecule(s) as it is credited with compounds having remarkable pharmacological properties. Recognizing the risk of loss of genetic variability of C. amada in response to imprudent exploitation of the natural resources coupled with unsystematic cultivation practices, preservation of its genetic resources has attracted the current research attention. The present study exploited the multifarious properties of Luffa sponge (i.e. sound physical strength, stability in texture/shape over wide pH range and repeated autoclaving, cost effective availability, etc.) as a novel matrix for synthetic seed conservation. In this study, we have evaluated the consequences of the presence of two fungicides, namely - bavistin and rosebengal, in the gel matrix as well as in the Luffa sponge for conservation of contamination free germplasm of C. amada. A maximum of 50% and 75% regrowth could be recorded amongst rosebengal and bavistin containing synthetic seeds respectively after 4 weeks of plating on plant growth regulator-free MS medium. Further growth of the encapsulated microshoots was significantly impeded by the presence of rosebengal within the synthetic seeds. On the contrary, the bavistin containing synthetic seeds demonstrated five times better regeneration ability to contamination-free shoots. A total of 78% survival was achieved upon transfer of these healthy plantlets to glass-house. RAPD fingerprinting revealed 84.62% genetic similarity between randomly selected synthetic seed derived plantlets. This report strengthened the vital conservation approach of C. amada using inexpensive Luffa sponge as storage matrix and bavistin for eradication of contaminations.     

Status of Bactrocera invadens (Diptera: Tephritidae) in Mango-Producing Areas of Arba Minch,Southwestern Ethiopia

Bactrocera invadens, the Asian fruit fly, was first reported in Kenya in 2003, and it spread fast to most tropical countries in Africa. To our knowledge, there is no detailed data on the fruit damage and status of fruit flies in Arba Minch and elsewhere in Ethiopia. Hence, information on the species composition and pest status of the fruit fly species is urgent to plan management strategies in the area. Fruit flies were captured using male parapheromone-baited traps. Matured mango (Mangifera indica) fruits were collected from randomly selected mango trees and incubated individually in cages (15 by 15 by 15 cm) with sandy soil. B. invadens was the predominant (96%; 952 of 992) captured species and the only fruit fly species emerging from mango fruits incubated in the laboratory. The mean number of adult B. invadens emerging per mango fruit was 35.25, indicating that the species is the most devastating mango fruit fly in the area. The loss due to this species would be serious if no management strategies are implemented.