Population structure of the predatory mite <Emphasis Type="Italic">Neoseiulus womersleyi</Emphasis> in a tea field based on an analysis of microsatellite DNA markers

The predatory mite Neoseiulus womersleyi (Schicha) (Acari: Phytoseiidae) is an important natural enemy of the Kanzawa spider mite, Tetranychus kanzawaki Kishida (Acari: Tetranychidae), in tea fields. Attraction and preservation of natural enemies by habitat management to reduce the need for acaricide sprays is thought to enhance the activity of N. womersleyi. To better conserve N. womersleyi in the field, however, it is essential to elucidate the population genetic structure of this species. To this end, we developed ten microsatellite DNA markers for N. womersleyi. We then evaluated population structure of N. womersleyi collected from a tea field, where Mexican sunflower, Tithonia rotundifolia (Mill.), was planted to preserve N. womersleyi. Seventy-seven adult females were collected from four sites within 200 m. The fixation indexes F _ST among subpopulations were not significantly different. The kinship coefficients between individuals did not differ significantly within a site as a function of the sampling dates, but the coefficients gradually decreased with increasing distance. Bayesian clustering analysis revealed that the population consisted of three genetic clusters, and that subpopulations within 100 m, including those collected on T. rotundifolia, were genetically similar to each other. Given the previously observed population dynamics of N. womersleyi, it appears that the area inhabited by a given cluster of the mite did not exceed 100 m. The estimation of population structure using microsatellite markers will provide valuable information in conservation biological control.     

Phylogenetic Analysis of the Spider Mite Sub-Family Tetranychinae (Acari: Tetranychidae) Based on the Mitochondrial COI Gene and the 18S and the 5′ End of the 28S rRNA Genes Indicates That Several Genera Are Polyphyletic

The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp). As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered.     

Phytoseiid mite species composition in Japanese peach orchards estimated using quantitative sequencing

We attempted a population survey of spider mites and phytoseiid mites in Japanese peach orchards with different pesticide practices; however, we had difficulty discriminating phytoseiid mites. To estimate phytoseiid mite species composition, ribosomal gene fragments were amplified from genomic DNA of five phytoseiid mite species using PCR. Cloning and nucleotide sequencing of amplified fragments identified species-specific polymorphic sites. Newly amplified fragments from recombinant plasmids were mixed in various ratios to produce standard DNA template mixtures. After direct sequencing, the signal ratios between two nucleotides at each species-specific polymorphic site were calculated and shown against the corresponding expected ratios. Quadratic regression equations were used to estimate the phytoseiid mite species composition. Results showed that the phytoseiid mite species composition changed during the survey period and varied among study sites.     

A novel method for protecting slow-release sachets of predatory mites against environmental stresses and increasing predator release to crops

Slow-release sachets of predatory mites are widely employed for controlling small pest arthropods in protected crops. However, environmental stresses can adversely affect the performance of such sachets. To solve this problem, we developed plant-attached shelters that hold sachets of Neoseiulus californicus (McGregor) or Amblyseius swirskii (Athias-Henriot). We conducted laboratory experiments to reveal whether sheltered sachets can protect predators against pesticides and drenching. The numbers of each predator in unsheltered sachets were drastically decreased after spraying with a non-selective pesticide (methidathion), and after continuous spraying (four days) with water, whereas the numbers in the sheltered sachets were not seriously affected by these factors. We also found that more predators (at least for N. californicus) were released from sheltered sachets at different temperatures (25 and 17 °C) than from unsheltered sachets. These results indicate sheltered sachets to be potentially useful for protecting predatory mites against environmental stresses and enhancing their release to crops.     

Phenotypic Characterization of the Komeda Miniature Rat Ishikawa, an Animal Model of Dwarfism Caused by a Mutation in Prkg2

The Komeda miniature rat Ishikawa (KMI) is a spontaneous animal model of dwarfism caused by a mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII). This strain has been maintained as a segregating inbred strain for the mutated allele mri. In this study, we characterized the phenotype of the KMI strain, particularly growth traits, craniofacial measurements, and organ weights. The homozygous mutant (mri/mri) animals were approximately 70% to 80% of the size of normal, heterozygous (mri/+) animals in regard to body length, weight, and naso-occipital length of the calvarium, and the retroperitoneal fat of mri/mri rats was reduced greatly. In addition, among progeny of the (BN×KMI-mri/mri)F1×KMI-mri/mri backcross, animals with the KMI phenotype (mri/mri) were easily distinguished from those showing the wild-type phenotype (mri/+) by using growth traits such as body length and weight. Genetic analysis revealed that all of the backcrossed progeny exhibiting the KMI phenotype were homozygous for the KMI allele in the 1.2-cM region between D14Rat5 and D14Rat80 on chromosome 14, suggesting strongly that mri acts in a completely recessive manner. The KMI strain is the first and only rat model with a confirmed mutation in Prkg2 and is a valuable model for studying dwarfism and longitudinal growth traits in humans and for functional studies of cGKII.Abbreviations: cGKII, cGMP-dependent protein kinase type II; CNP, C-type natriuretic peptide; KMI, Komeda miniature rat IshikawaDwarfism is caused by both endocrinologic and nonendocrinologic defects. Most instances of dwarfism, including normal variants, are nonendocrinologic, and subjects retain growth hormone secretion. Although spontaneous rodent models of dwarfism with confirmed mutations have been reported—Snell dwarf mice with Pou1f1 (Pit1) mutation,¹⁴ Ames dwarf mice with Prop1 mutation,²² little mice with Ghrhr mutation,¹⁵ pygmy mice (also known as mini-mice) with Hmga2 (HMGI-C) mutation,²⁶ spontaneous dwarf rats with Gh mutation,²³ and rdw rats with Tg mutation^9,11—most of these are models of endocrinologic dwarfism. A few models of nonendocrinologic dwarfism have been produced by gene manipulation techniques, such as transgenic and knockout strategies, and include Col2a1-transgenic mice,^7,24 Col10a1-transgenic mice,¹⁰ and Fgfr3-knock-in mice.¹³A novel spontaneous dwarf mutation, miniature rat Ishikawa (mri), was discovered in a closed colony of Wistar rats at Ishikawa Animal Laboratory (Saitama, Japan) and has been maintained on the genetic background of Wistar rats. This mutant strain, previously termed Miniature Rat Ishikawa (MRI), has recently been established as a segregating inbred strain on the Wistar genetic background, designated Komeda Miniature rat Ishikawa (KMI). The breeding record suggested that the mutation was inherited in an autosomal recessive mode. KMI rats show no abnormality in the basal amounts or distribution of several hormones, including growth hormone, luteinizing hormone, follicle-stimulating hormone, prolactin, thyroid-stimulating hormone, and adrenocorticotropic hormone, but growth hormone response to growth hormone releasing hormone is decreased.²¹Using positional candidate cloning of mri, we recently identified a deletion mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII), and clarified a role of cGKII as a molecular switch that couples cessation of proliferation and the start of hypertrophic differentiation of chondrocytes.² Longitudinal skeletal growth is achieved by endochondral ossification in the growth plate, in which chondrocyte hypertrophic differentiation is an important step. Due to the impaired coupling of proliferation and hypertrophic differentiation in the growth plate chondrocytes, homozygous mutant (mri/mri) animals show longitudinal growth retardation.In this study, we further characterize the phenotype of the KMI strain, including body length, body weight, organ weight, and craniofacial measurements. Furthermore, we describe phenotypic characteristics of the progeny produced from the (BN×KMI-mri/mri)F1×KMI-mri/mri backcross and provide updated genetic, physical, and comparative maps of the mri region.     

Optimized aeration by carbon dioxide gas for microalgal production and mass transfer characterization in a vertical flat-plate photobioreactor

To optimize the aeration conditions for microalgal biomass production in a vertical flat-plate photobioreactor (VFPP), the effect of the aeration rate on biomass productivity was investigated under given conditions. Air enriched with 5% or 10% (v/v) CO(2) was supplied for the investigation at rates of 0.025-1 vvm. The CO(2) utilization efficiency, change of pH in the medium, and the optimum aeration rate were determined by evaluating biomass productivity. To investigate the VFPP mass transfer characteristics, the overall volumetric mass transfer coefficient, k(L)a, was evaluated for several different flat-plate sizes. Increasing the height of the VFPP could improve both the mass transfer of CO(2) and the illumination conditions, so this appeared to be a good method for scaling up. Based on a comparison of the k(L)a value at the optimum aeration rate with previously reported results, it was confirmed that the range of CO(2) concentration used in the experiments was cost-effective for mass culture.     

Isolation, characterization, inheritance and linkage of microsatellite markers in Tetranychus kanzawai (Acari: Tetranychidae)

We isolated and characterized seven microsatellite markers in Tetranychus kanzawai (Acari: Tetranychidae). We also examined the conformity of the isolated markers to Mendelian laws and analyzed linkage among the microsatellite loci. All microsatellite markers fit expected 1:1 disomic segregation ratio and hence were inherited in a Mendelian manner. Significant pairwise linkage was detected in three pairs of microsatellite loci. These isolated microsatellite markers may become a powerful tool for the study of behavioral ecology, population genetics, and genome mapping of T. kanzawai.     

Microalgal studies for the 21st Century

Microalgal photosynthesis is efficient enough to fix CO₂ in both atmosphere and industrially discharged gases, and is a possible future alternative for CO₂ reduction. This paper describes physiological responses of microalgal cells to extremely high CO₂ concentrations, capability of microalgal cells to fix CO₂ at both indoor and outdoor culture experiments, and efforts to establish a culture collection of marine microalgae. Recent researches indicate that microalgae are likely to play a key role in worldwide issues of the coming century.     

Restriction fragment length polymorphism catalog for molecular identification of Japanese Tetranychus spider mites (Acari: Tetranychidae)

Species identification is a basic issue in biosecurity. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) is a useful molecular diagnostic tool for species identification. However, the lack of transferability of data has been a serious shortcoming of this method. A RFLP catalog, i.e., a graph of PCR-RFLP patterns expected from sequence data, was devised as a tool to facilitate PCR-RFLP data sharing among laboratories. Twelve species of Tetranychus spider mites have been recorded in Japan to date. In this study, we analyzed DNA sequences of the internal transcribed spacer (ITS) region in nuclear ribosomal DNA of 11 Tetranychus species. For the species identification using PCR-RFLP, we chose six candidates from 131 restriction endonucleases and developed an RFLP catalog of all known Japanese Tetranychus species except Tetranychus neocaledonicus André. The RFLP catalog revealed that most Tetranychus species had diagnostic restriction fragments. The RFLP catalog is transferable and simple molecular diagnostic tool, and it has the ability to add more species and newly found intraspecific variations. Therefore, we believe that the RFLP catalog will contribute to biosecurity as a practical diagnostic tool for species identification of spider mites.