Simultaneous identification of Grapholita molesta Busck and Grapholita dimorpha Komai by PCR‐RFLP

Although several molecular diagnostic techniques are available for the identification of the apple‐feeding pests Grapholita molesta Busck and Grapholita dimorpha Komai, these pests are severely affecting apple orchards in Korea. These two pests may be misidentified or the available molecular diagnostic techniques may not facilitate the simultaneous identification of the morphological features of both species. In this study, we developed a multiplex assay for these two species using the polymerase chain reaction – restriction fragment length polymorphism (PCR‐RFLP) method. Sixty‐two specimens were collected from apples presumed infested with moth larvae and from pheromone traps from 2013 to 2014. Both species were identified morphologically, and a partial region of the cytochrome b gene was sequenced to design primers for PCR‐RFLP. Digestion profiles of G. molesta and G. dimorpha, using the Sau3A1 restriction enzyme, were characterized using three DNA fragments each for G. molesta (363 bp, 91 bp and 31 bp) and G. dimorpha (220 bp, 234 bp and 31 bp). The RFLP assay developed for both species in this study was more efficient and accurate than other currently used diagnostic assays and would be helpful to identify field‐collected specimens for pest control research.     

Model of Grapholita molesta spring emergence in pear orchards based on statistical information criteria

The oriental fruit moth, Grapholita molesta, is becoming a large threat to Korean pear production. Timely management of the egg and early larval stages from the spring emergence is critical to reduce the G. molesta population during the pear growing season. A model was developed to precisely predict the spring occurrence of G. molesta adults as a function of accumulated degree-days. The model was validated with male moth caught in sex pheromone-baited traps placed in pear orchards at two major pear production regions (Icheon and Naju) of Korea in 2010. We applied nine distribution models to describe the cumulative proportions of G. molesta males caught relative to accumulated degree-days. The observed phenology of the G. molesta spring population was well described by the nine models. The predicted dates for the cumulative 50% male moth catches were within a 5 day period. Based on statistical information criteria (Akaike's and Bayes–Schwartz information criteria), we recommend the sigmoid function referred by Brown and Mayer, because of its ease of use and meaningfulness; the parameter “b” denotes the degree-day accumulation at 50% moth emergence. The G. molesta spring emergence model could be applied to determine optimal chemical treatment timing for controlling G. molesta in fruit tree orchards and further help to develop a full-cycle phenology model of G. molesta.     

Microsatellite loci for Australian agamid lizards

We characterize 15 microsatellite loci from two microsatellite libraries developed from the Australian agamid lizards Amphibolurus muricatus and Ctenophorus pictus. All loci were tested for amplification in four other agamids: Ctenophorus fordi, Ctenophorus decresii, Chlamydosaurus kingii, and Physignathus lesueurii. These loci were highly polymorphic within and across species, with nine to 12 loci amplifying for each species tested.     

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.     

Microsatellite markers for the fungal banana pathogens Mycosphaerella fijiensis, Mycosphaerella musicola and Mycosphaerella eumusae

We developed a total of 50 microsatellite markers for the three fungal pathogens causing the most important leaf spot diseases of banana: 32 loci for Mycosphaerella fijiensis are presented, and nine loci each for Mycosphaerella musicola and Mycosphaerella eumusae. All these loci were polymorphic within each species on a sample of isolates collected from various locations around the world. Within M. fijiensis and M. musicola, most of the loci tested (> 80%) in a sample of isolates from a single location in Cameroon were also polymorphic. Multiplex polymerase chain reaction systems were developed with 15 loci for M. fijiensis.     

High Genetic Diversity and Structured Populations of the Oriental Fruit Moth in Its Range of Origin

The oriental fruit moth Grapholita ( = Cydia) molesta is a key fruit pest globally. Despite its economic importance, little is known about its population genetics in its putative native range that includes China. We used five polymorphic microsatellite loci and two mitochondrial gene sequences to characterize the population genetic diversity and genetic structure of G. molesta from nine sublocations in three regions of a major fruit growing area of China. Larval samples were collected throughout the season from peach, and in late season, after host switch by the moth to pome fruit, also from apple and pear. We found high numbers of microsatellite alleles and mitochondrial DNA haplotypes in all regions, together with a high number of private alleles and of haplotypes at all sublocations, providing strong evidence that the sampled area belongs to the origin of this species. Samples collected from peach at all sublocations were geographically structured, and a significant albeit weak pattern of isolation-by-distance was found among populations, likely reflecting the low flight capacity of this moth. Interestingly, populations sampled from apple and pear in the late season showed a structure differing from that of populations sampled from peach throughout the season, indicating a selective host switch of a certain part of the population only. The recently detected various olfactory genotypes in G. molesta may underly this selective host switch. These genetic data yield, for the first time, an understanding of population dynamics of G. molesta in its native range, and of a selective host switch from peach to pome fruit, which may have a broad applicability to other global fruit production areas for designing suitable pest management strategies.     

Development, characterization and transferability of microsatellite markers for the plant genus Phlox (Polemoniaceae)

In order to study diversification and microevolution in Phlox, we developed nine polymorphic microsatellite loci. In 20 individuals of Phlox pilosa from a single population, the average number of alleles per locus was 10.0 ± 5.1, and average observed and expected heterozygosities were 0.611 ± 0.234 and 0.769 ± 0.170, respectively. Most of these markers amplified successfully in 11 additional species of Phlox, representing a broad diversity of the genus, and some also amplified in more distantly related members of the Polemoniaceae. These microsatellite markers will be valuable for investigation of evolutionary processes in this important study system.     

Strong correlation between cross-amplification success and genetic distance across all members of 'True Salamanders' (Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite loci

The unpredictable and low cross-amplification success of microsatellite loci tested for congeneric amphibian species has mainly been explained by the size and complexity of amphibian genomes, but also by taxonomy that is inconsistent with phylogenetic relationships among taxa. Here, we tested whether the cross-amplification success of nine new and 11 published microsatellite loci cloned for an amphibian source species, the fire salamander (Salamandra salamandra), correlated with the genetic distance across all members of True Salamanders (genera Chioglossa, Lyciasalamandra, Mertensiella and Salamandra that form a monophyletic clade within the family of Salamandridae) serving as target species. Cross-amplification success varied strongly among the species and showed a highly significant negative relationship with genetic distance and amplification success. Even though lineages of S. salamandra and Lyciasalamndra have separated more than 30 Ma, a within genus amplification success rate of 65% was achieved for species of Lyciasalamandra thus demonstrating that an efficient cross-species amplification of microsatellite loci in amphibians is feasible even across large evolutionary distances. A decrease in genome size, on the other hand, paralleled also a decrease in amplified loci and therefore contradicted previous results and expectations that amplification success should increase with a decrease in genome size. However, in line with other studies, our comprehensive dataset clearly shows that cross-amplification success of microsatellite loci is well explained by phylogenetic divergence between species. As taxonomic classifications on the species and genus level do not necessarily mirror phylogenetic divergence between species, the pure belonging of species to the same taxonomic units (i.e. species or genus) might be less useful to predict cross-amplification success of microsatellite loci between such species.     

Isolation and characterization of microsatellite loci from three cryptic species of Ceratosolen emarginatus

From an enriched (AG)_n/(AC)_n library, we developed nine polymorphic microsatellite loci for three cryptic species of the fig‐pollinating wasp Ceratosolen emarginatus. We genotyped one population for each of the three cryptic species across all the nine loci. In total, 204 alleles were detected from the three cryptic species of C. emarginatus. The observed heterozygosity was 0.755 ± 0.034, 0.653 ± 0.030 and 0.603 ± 0.073 in C. emarginatus populations A, B and C, respectively; the expected heterozygosity was 0.850 ± 0.031, 0.724 ± 0.035 and 0.702 ± 0.104, respectively. No linkage disequilibrium was found between any two loci of all three cryptic species. The newly isolated microsatellite markers will be very useful for estimating the genetic variation within and among the cryptic species and for revealing the mechanisms of speciation and inbreeding coexistence hypothesis of the cryptic species.     

Eleven microsatellite loci in the sociobiologically enigmatic ant Lasius austriacus (Hymenoptera: Formicidae)

We describe the isolation and characterization of 11 microsatellite loci in the sociobiologically enigmatic ant Lasius austriacus. The polymerase chain reaction primers were tested on a population in East Austria. The number of alleles ranged from four to 19 and the observed heterozygosity from 0.200 to 0.900. Cross‐species amplification tests were performed, with some loci polymorphic in all species tested, for the closely related invasive species Lasius neglectus, three further Lasius species, another formicine, Formica polyctena, and the invasive myrmicine species Tetramorium tsushimae.     

Development of 10 polymorphic microsatellite loci isolated from the mountain beaver, Aplodontia rufa rufa (Rafinesque)

We developed 10 microsatellite markers for the mountain beaver, Aplodontia rufa rufa. In three populations of A. r. rufa, the number of alleles for these loci ranged from monomorphic to nine. Average observed heterozygosities in these populations ranged from 0.29 to 0.60. We also tested previously published markers from the endangered subspecies A. r. nigra in A. r. rufa populations.     

Microsatellites and microsynteny in the chloroplast genomes of Oryza and eight other Gramineae species

Primer pairs flanking ten chloroplast microsatellite loci, originally identified in Oryza sativa cv Nipponbare, were evaluated for amplification and allelic diversity using a panel of 13 diverse cultivars of rice (O. sativa), 19 accessions of wild rice (three O. officinalis, five O. latifolia, five O. minuta, four O. australiensis, one O. brachyantha and one O. ridleyi) and eight other Gramineae species (maize, teosinte, wheat, oat, barley, pearl millet, sorghum and sugarcane). Amplified products were obtained for all samples at nine out of ten loci. Among the rice cultivars, the number of alleles per locus ranged from one to four, with monomorphic patterns observed at five loci. The average polymorphism information content (PIC) value at the other five (polymorphic) loci was 0.54 among the 13 cultivars. When wild rice and the other Gramineae species were compared based on the proportion of shared alleles, their phylogenetic relationships were in agreement with previous studies using different types of markers; however, the magnitude of the differences based on chloroplast microsatellites underestimated the genetic distance separating these divergent species and genera. A sequence-based comparison of homologous regions of the rice and maize chloroplast genomes revealed that, while a high level of microsynteny is evident, the occurrence of actively evolving microsatellite motifs in specific regions of the rice chloroplast genome appears to be mainly a species or genome-specific phenomenon. Thus the chloroplast primer pairs used in this study bracketed mutationally active microsatellite motifs in rice but degenerate, interrupted motifs or highly conserved, mutationally inert motifs in distantly related genera. Received: 17 March 1999 / Accepted: 11 November 1999     

Characterization of nine microsatellite loci in the sea star Astropecten aranciacus and cross-species amplification for related taxa

So far, only few microsatellite markers have been developed and extensively tested for echinoderms. To study the population genetic structure of the sea star Astropecten aranciacus, we developed primers for nine polymorphic microsatellite loci and tested them on two populations from Faro in Portugal (N = 25) and from La Herradura in Spain (N = 20). Within populations, allele numbers varied from four to 20, while expected and observed heterozygosities ranged from 0.593 to 0.936 and from 0.222 to 0.900, respectively. Additional cross‐species amplifications were polymorphic at some loci, indicating their potential usefulness for related taxa.     

Olfactory responses of Grapholita molesta to different developmental stages of immature fruit of peach,plum, and apple

The Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is known to shift its host use from stone fruits (the primary hosts) to pome fruits (secondary hosts) as the season progresses. Grapholita molesta recognizes potential hosts by semiochemicals from fruits, but few studies have addressed the olfactory responses of G. molesta to host fruits during the seasonal transition of the moth from stone to pome fruits. So, we investigated the olfactory responses of larvae and gravid females of G. molesta to different fruit stages (collected on May 31, June 11, and June 25) of immature peach, plum, and apple fruits, using both multiple and two choice tests. In the multiple choice tests, for fruits collected after June 11, larvae showed a significant preference for peach as their first choice, compared to plum or apple. The final choice rate (when larvae had stayed on the fruit for more than 1 h) of larvae was also highest for peach fruits collected on June 11 and June 25. In two choice tests (Y-tube test), the response rate and overall choice rate of larvae and mated ovipositing adult females were also highest on peaches collected on May 31 (larvae) and June 25 (adults), respectively. In conclusion, we found that both the adults and larvae of G. molesta were highly attracted to immature peach fruits in laboratory choice tests.     

A diagnostic multiplex polymerase chain reaction method to identify Japanese internal apple-feeding Lepidopteran pests: Grapholita molesta, Grapholita dimorpha (Lepidoptera: Tortricidae), and Carposina sasakii (Lepidoptera: Carposinidae)

We present a multiplex polymerase chain reaction method to differentiate between three Japanese internal apple-feeding pests: the oriental fruit moth, Grapholita molesta (Busck), Grapholita dimorpha Komai, and the peach fruit moth, Carposina sasakii Matsumura. A 1,342-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in each species. The three species showed consistent and diagnostic differences in the region of the COI gene, from which three species-specific forward primers were designed. The forward primers along with one universal reverse primer were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. This method enabled easy, immediate, and accurate identification of internal feeding Lepidoptera in apples and other fruits.     

Field observations quantifying attraction of four tortricid moths to high-dosage pheromone dispensers in untreated and pheromone-treated orchards

Orientational responses of four species of feral tortricid moths (Lepidoptera: Tortricidae) to polyethylene tube dispensers of pheromone were observed in a 0.8 ha apple orchard treated with such pheromone dispensers and in an untreated 0.8 ha orchard. Male oblique‐banded leafrollers, Choristoneura rosaceana (Walker) (mean 7.2 ± 0.4 moths/night during 21 nights), Oriental fruit moths, Grapholita molesta (Busck) (mean 10.5 ± 2.1 during 20 evenings), and the redbanded leafrollers, Argyrotaenia velutinana (Walker) (mean 2.0 ± 1.1 during 14 nights) were observed approaching within 100 cm of their respective polyethylene‐tube pheromone dispensers in the untreated orchard. Furthermore, C. rosaceana (mean 2.0 ± 0.7 during 17 nights) and G. molesta (mean 1.5 ± 0.4 over 20 evenings) came within 100 cm of their respective polyethylene‐tube pheromone dispensers in the pheromone‐treated orchard. Most visits lasted less than 10 s, after which the majority of moths departed by flying upwind. In the untreated orchard, the number of C. rosaceana observed orienting to polyethylene tube dispensers was greater than the number captured in optimized monitoring traps (1.9 ± 0.4) per night of observation. The numbers of A. velutinana (2.0 ± 1.1) or G. molesta (10.5 ± 2.1) attracted to polyethylene‐tube dispensers in the untreated orchard did not differ statistically from the numbers captured in optimized monitoring traps per night of observation. In the pheromone‐treated orchard, the number of C. rosaceana (2.0 ± 0.4) or G. molesta (1.2 ± 0.2) observed orienting to polyethylene‐tube dispensers did not differ statistically from the numbers of male moths of these species captured in optimized monitoring traps per night of observation. No codling moths, Cydia pomonella L. were observed orienting to, or landing near, their respective polyethylene‐tube dispensers in either the untreated or pheromone‐treated orchards, although substantial numbers were captured in monitoring traps per night of observation (6.0 ± 1.7) in the untreated orchard. The attraction of male moths to polyethylene tube dispensers thus occurred in three of the four species observed. These results provide support for the idea that false‐plume‐following is an important component of the mechanisms mediating communicational disruption in moths by polyethylene‐tube dispensers.     

Isolation and characterization of polymorphic microsatellite loci in the field vole,Microtus agrestis,and their cross‐utility in the common vole,Microtus arvalis

We developed nine polymorphic microsatellite loci for the field vole, Microtus agrestis. The number of alleles ranged from five to 15 and observed heterozygosities ranged from 0.40 to 1.00. We also tested the microsatellite loci for amplification and polymorphism in the congeneric species Microtus arvalis. Five of the nine loci were successfully analysed in this species. The microsatellite markers will be employed in studies of reproductive success and fine‐scale spatial genetic structure.     

Thirteen polymorphic microsatellite DNA loci from whiptails of the genus Aspidoscelis (Teiidae: Squamata) and related cnemidophorine lizards

We describe polymerase chain reaction primers and amplification conditions for 13 microsatellite DNA loci isolated from two bisexual species of whiptail lizards Aspidoscelis costata huico and Aspidoscelis inornata. Primers were tested on either 16 or 48 individuals of A. c. huico and/or 26 individuals of A. inornata. Ten of the 13 primers were also tested against a panel of 31 additional whiptail taxa. We detected three to nine alleles per locus in A. c. huico and four to 19 alleles per locus in A. inornata, with observed heterozygosity ranging from 0.60 to 0.87 and from 0.15 to 1.00, respectively. These primers will be an important resource for surveys of genetic variation in these lizards.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of obscure puffer (Takifugu obscurus) and cross-species amplification

Obscure puffer (Takifugu obscurus) is an anadromous fish species in China. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of T. obscurus. The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from four to 10, from 0.57 to 0.86 and from 0.68 to 0.90, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in T. obscurus.