Improvement of DNA extraction method for dried blood spots and comparison of four PCR methods for detection of Babesia gibsoni (Asian genotype) infection in canine blood samples

To eradicate canine babesiosis in epidemic areas, mass-screening of the infection situation of Babesia gibsoni including occult infection is necessary. The development of cost-effective method for storage and transport of blood samples is required. A highly efficient DNA extraction procedure from dried blood spots (DBS) onto Whatman 3MM filter paper was developed for the diagnosis of B. gibsoni infection in dog by PCR. In 3 extraction methods, Chelex-based method in combination with saponin washing and phenol-chloroform-isoamyl alcohol extraction (Saponin-PCI method) provided the best results. Sensitivity of the 4 previously described PCR methods for detection of B. gibsoni infection was also compared using serially diluted blood samples of B. gibsoni-infected dogs. The PCR method using Gib599F/Gib1270R primer pair provided the best performance. To evaluate the stability of DNA in DBS, DBS of B. gibsoni-infected dogs stored at room temperature for 2 months. The stability was superior to whole blood samples stored at -20 degrees C for 2 months. This highly efficient DNA extraction method on DBS using Whatman 3MM filter paper has potential to be cost-effective and high performance tool for storage, and molecular diagnosis of clinical blood sample from dog. This procedure in combination with the PCR method using Gib599F/Gib1270R primer pair may greatly assist in diagnosis of B. gibsoni infection in dog populations that are geographically distant.     

Breed differentiation among Japanese native chickens by specific skull features determined by direct measurements and computer vision techniques

1. Inter-breed morphological comparisons were made among 11 breeds of Japanese native chickens (Gifujidori, Hinaidori, Shokoku, Totenko, Tomaru, Satsumadori, Shamo, Koshamo, Koeyoshi, Chabo and Nagoya), White Leghorn, broiler chickens (Chunky) and red junglefowl collected in the Philippines, based on results of direct measurements and analysis by computer vision techniques of the skull. 2. Analysis of direct measurements identified two groups of chicken: a small type that included the Chabo, Koshamo, red junglefowl, Gifujidori and Shokoku and a large type that included the remaining breeds studied. These groupings were made based on size determined both in the first (PC1) and second principal component (PC2). The greatest length of the cranium and condylobasal length greatly contributed to the morphological differences between these two groups. 3. Analysis by computer vision techniques, however, identified three groups: the Bantam group (which includes red junglefowl), Shokoku group and Shamo group. White Leghorn clustered within the Shokoku group while the broiler chicken belonged to the Shamo group. The region around the junction of the neural cranium and the visceral cranium contributed greatly to the morphological differences among breeds, both in the PC1 and PC2.     

Anther culture in rice proportionally rescues microspores according to gametophytic gene effect and enhances genetic study of hybrid sterility

Background

To investigate plant hybrid sterility, we studied interspecific hybrids of two cultivated rice species, Asian rice (Oryza sativa) and African rice (O. glaberrima). Male gametes of these hybrids display complete sterility owing to a dozen of hybrid sterility loci, termed HS loci, but this complicated genetic system remains poorly understood.

Results

Microspores from these interspecific hybrids form sterile pollen but are viable at the immature stage. Application of the anther culture (AC) method caused these immature microspores to induce callus. The segregation distortion of 11 among 13 known HS loci was assessed in the callus population. Using many individual calli, fine mapping of the HS loci was attempted based on heterozygotes produced from chromosome segment substitution lines (CSSLs). Transmission ratio distortion (TRD) from microspores was detected at 6 of 11 HS loci in the callus population. The fine mapping of S₁ and S₁₉ loci using CSSLs revealed precise distances of markers from the positions of HS loci exhibiting excessive TRD.

Conclusions

We demonstrated that AC to generate callus populations derived from immature microspores is a useful methodology for genetic study. The callus population facilitated detection of TRD at multiple HS loci and dramatically shortened the process for mapping hybrid sterility genes.

     

Quantifying landscape pattern and ecosystem service value changes in four rapidly urbanizing hill stations of Southeast Asia

Context

Hill stations are known for their favorable cool climate and natural environments which generate valuable ecosystem services that benefit the local population, tourists and visitors. However, rapid urbanization threatens the sustainability of these highly valued fragile landscapes.

Objectives

We aim to characterize and quantify the changes in the landscape patterns and ecosystem service values (ESVs) of Baguio (Philippines), Bogor (Indonesia), Dalat (Vietnam), and Pyin Oo Lwin (Myanmar), and discuss their implications to landscape sustainability.

Methods

We used remote sensing imagery to map land-use/cover (2001 and 2014), and spatial metrics and gradient analysis to characterize the changes in landscape pattern. We employed a benefit transfer method to estimate the changes in ESV and human-to-ESV ratio. A land-change model was used to simulate different scenarios of future built-up expansions (2014–2030).

Results

The landscapes of Dalat and Pyin Oo Lwin are becoming more fragmented, while those of Baguio and Bogor are getting more aggregated. Dalat had the highest decrease (absolute change) in ESV and H-ESV ratio, while Bogor had the highest percentage decrease (2001–2014).

Conclusions

Rapid urbanization has been a major factor in the landscape transformation of Baguio, Bogor, Dalat and Pyin Oo Lwin. If the current built-up expansion rate will speed up, the decline in future ESV and H-ESV ratio (2014–2030) will be higher than if the rate will continue or slow down. Unless the concept of landscape sustainability is taken seriously in landscape and urban planning, the respective ‘values’ of these precious hill stations will become less and less.

     

Detection of genetic variation among seed and seedlings ofChamaecyparis obtusa using allozyme markers

The effects of nursery practices on genetic composition and structure were studied inChamaecyparis obtusa by analyzing seeds and one-, two-, and three-year-old seedlings. Enzyme polymorphisms of the6Pgd-2, G6pd, Got, Gk, Shd-2, Pod, Dia-1, andPgm loci were used. OnlyDia-1 indicated significantly different allelic frequencies between seeds and three-year-old seedlings. Most of the genetic diversity of the parental clones in this seed orchard could be transferred to the offsprings. Genetic diversity on the basis of an average expected heterozygosity did not differ between seeds and seedlings. A slight excess of homozygotes in seeds and a significant excess of heterozygotes in seedlings of different ages to the expected panmictic proportions were found. Most of the differences in genotypic distributions among the seed and seedling stages, probably resulted from viability selection favoring outcrosses, and selective removal of inbred offsprings including self-fertilized ones prior to their transplantation in the nursery. A part of this paper was orally presented at the 107th Annual Meeting of the Japanese Forestry Society (1996).     

Liquid penetration of precompressed wood VI: Anatomical characterization of pit fractures

Pit fractures of refractory coniferous heartwoods caused by precompression in the radial direction were investigated and are discussed in terms of improved liquid penetration. Small cracks appeared at the boundary between the torus and margo, along the outer margin of the margo, and on the torus when specimens were compressed and deformation was fixed by drying. The remarkable cracks were generally observed for Cryptomeria japonica D. Don. Pseudotsuga menziesii Franco showed peculiar detachment of the torus from the pit border, and Larix leptolepis Gordon exhibited only small cracks on the torus. These fractures patterns were clearer when the precompressed specimens were recovered by water impregnation and then redried.This report was presented at the 46th Annual Meeting of the Japan Wood Research Society, Kumamoto, April 1996     

Immunochemical sexing of living yellowfin tuna, Thunnus albacares (Bonnaterre), using a vitellogeninlike protein

Sexing of living yellowfin tuna, Thunnus albacares (Bonnaterre), was carried out based on immunochemical detection of vitellogenin (VTG), a female-specific protein. Tissue samples were taken from swimming fish in a sea pen by a hand-made apparatus. Recently fertilized eggs were also collected before tissue sample trials. A double immunodiffusion pattern showed that the egg homogenate of T. albacares reacted with rabbit antibody against the purified VTG of greater amberjack, Seriola dumerili (Risso). This suggests that the VTG of T albacares has an antigenicity similar to that of S, dumerili and that this antibody can be used to detect T albacares VTG. Immunodot-blotting analysis revealed that, out of the two fish tested, the tissue sample from one, but not the other, reacted with this antibody, suggesting that the former was a mature female, and the latter a male or immature female. Examination of the fish gonads revealed an ovary and a testis, respectively. Immunological and morphological results corresponded completely. Therefore, the method used in the present study is available for discriminating the sexes of T albacares.     

Value-per-recruit analysis of bighand thornyhead <Emphasis Type="Italic">Sebastolobus macrochir</Emphasis> caught off the Pacific coast of northern Honshu,Japan

The yield-per-recruit (YPR) analysis has been used to assess growth overfishing of exploited fish stocks. Since the ex-vessel price per weight widely depends on the size of the fish, the result of the YPR does not imply the revenue. The value-per-recruit (VPR) analysis, which is an economic expansion of the YPR analysis, was conducted for bighand thornyhead Sebastolobus macrochir caught off the Pacific coast of northern Honshu, Japan. The results showed that, for the current age at first capture (t _c = 3), the current fishing mortality (F = 0.12) was close to the optimum level. However, for the current F, optimum t _c in the VPR model was 8 years and would lead to a 47% increase compared to the current revenue. The maximum YPR was obtained at t _c = 6 years, but the revenue at t _c = 6 only gave a 20% increase in the maximum revenue. The difference was caused by the size dependency of the ex-vessel price per weight. The VPR model would generate fisheries management plans with both economical and biological consideration and would help in making general consensus of fisheries management strategies.     

Assessing sexual maturity of feminized Japanese eel Anguilla japonica by measuring eye size

For efficient production of Japanese eel Anguilla japonica eggs, knowledge of the status of the sexual maturity of potential broodstock females is important because this status directly influences the time required to produce mature eggs by successive hormone doses. Here we apply an eye index (relative eye size) to evaluate the gonadal status of feminized A. japonica, which were induced by administration of estradiol-17β. Examination of gonad somatic and eye indexes of 267 feminized eels, cultivated for 12–56 months, revealed a significant correlation between these two indexes. Artificial maturation of 35 feminized eels showed that the number of hormone injections administered before ovulation was significantly lower as the eye index score increased, indicating availability of this noninvasive method of predicting sexual maturity of feminized eel. There was no correlation between eye index and fertilization rate, hatching rate, or larval survival rate, suggesting that sexual maturity before hormonal treatment does not affect egg quality.