A rapid and simple procedure for the whole-mount bone staining of small fish.

The cartilage and bone structure has provided insightful knowledge about evolution and ecology of fish, which is an important component of biological oceanography. However, the whole-body bone staining is a lengthy and complicated process that typically takes five days to several months, and the improvement of the conventional method has been one of the important issues in this field. Here we report a quick and easy whole-mount bone staining method for small fish, in which a newly designed fixative is applied. Compared to conventional methods, this novel protocol is a straightforward process that could be adopted for small estuarine fish and other small vertebrates.

Comprehensive Structure of the Female Marine Water-Strider Asclepios annandalei Distant, 1915 from Pranburi River Estuary, Thailand: New Information for the Genus Asclepios.

The objective of this study was to describe the structure and histochemistry of the systemic organs in the female marine water-strider Asclepios annandalei from Pranburi river estuary, Thailand. Results from this study revealed for the first time that the integumentary system of this species consisted of three layers including epicuticle, exocuticle and endocuticle. The muscular system apparently contained only skeletal muscle along the body. In the urinary system, we observed well-developed Malpighian tubules, each of which was covered with the simple cuboidal epitheliums. These epitheliums also contained the secretory granules that were reacted positively with periodic acid Schiff (PAS). The digestive system of this species was composed of three distinct parts including foregut, midgut and hindgut. The respiratory system was composed of the respiratory organ, which was rarely found near the integument system. This organ was lined with a simple squamous epithelium. Two regions of nervous system, i.e., frontal ganglion connected to the eye structure and ventral nerve cord, were found. Each ganglion basically consisted of two layers, outer cortex and inner medullae. The outer cortex contained three types of cells, including neurosecretory cells, neuroglial cells and neurons. The cytoplasmic inclusion of neurosecretory cells contained secretory granules, which reacted positively with PAS, indicating the presence the glycoprotein. The neuroglia and neuron were also observed in the inner medullae layer. The female reproductive system (the ovarian structure, the reproductive tract and the accessory organ) of this gerrid species was seen under histological sections. The well-developed integument system and Malpighian tubule as well as the abundant respiratory organ is a characteristic of this species, which might be useful for the adaption to the estuarine condition.

Effects of Intramuscular Injections of Vitamins AD3E and C in Combination on Fertility, Immunity, and Proteomic and Transcriptomic Analyses of Dairy Cows during Early Gestation.

This research aimed to investigate the effects of the intramuscular injection of vitamins AD3E and C in combination immediately before the estrus synchronization program (the Ovsynch program) on conception and pregnancy rates, blood parameters, serum biochemical properties, immune systems, antioxidant parameters, and proteomic and transcriptomic analyses during early gestation in dairy cows. Forty nonlactating multiparous cows were randomly assigned to one of four treatments: (1) C: control with normal saline injection; (2) VAD3E: a single intramuscular injection (I/M) of vitamin AD3E; (3) VAD3EC: injection of both vitamins AD3E and C; (4) VC: a single dose of vitamin C. Blood and serum samples were taken immediately at day 0 (before AI), day 7, and day 14 (after AI for 5 days) from the coccygeal vein. Generally, injections of AD3E and C in combination had no effect on the rate of conception or pregnancy. However, they improved hematological parameters and immune and antioxidant activities. Serum samples were analyzed using LC-MS/MS, and 8190 proteins were identified. Five proteins were successfully validated using the quantitative real-time reverse transcription PCR (qRT-PCR) method. This study found that lymphocyte-specific protein 1 (LSP1, A0A3Q1M894) could be used as a protein biomarker for cows administrated with vitamins AD3E and C.

Cannabinoids from inflorescences fractions of Trema orientalis (L.) Blume (Cannabaceae) against human pathogenic bacteria.

BACKGROUND: Cannabinoids; tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN), might show antibacterial activity. Trema orientalis is a species in the Cannabaceae that is closely related to Cannabis through plastome phylogenetic evidence. This species is widely distributed throughout tropical Asia and is used as traditional medicine, particularly for the treatment of infectious diseases. However, no studies on the antibacterial activity of cannabinoid-containing inflorescences extracts are available. Thus, the aim of this study was to determine cannabinoid content and antibacterial activity of inflorescences fractions from T. orientalis native to Thailand. METHODS: We hypothesized that inflorescences from T. orientalis might display cannabinoids similar to Cannabis because of their close taxonomic relationship. We extracted the mature inflorescences and infructescence of T. orientalis in three disparate populations from different Thailand floristic regions. Extractions were subsequently partitioned into hydrophilic and lipophilic fractions using distilled water and chloroform. The lipophilic extracts were further fractionated by the column chromatography with gradient elution and analyzed by gas chromatography-mass spectrometry (GC-MS). Characterized cannabinoids were used in bioassays with multidrug-resistance bacteria. RESULTS: Lipophilic extracts and fractions of inflorescences from all Thailand floristic regions consistently displayed cannabinoids (THC, CBD and CBN) in various quantities. These extracts exhibited inhibitory activity for Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii strains with minimum inhibitory concentration values varying from 31.25 to 125 µg/mL. CONCLUSION: Our study is the first to report cannabinoid detection in extracts from inflorescences of T. orientalis, a species in the Cannabaceae. These extracts and their fractions containing cannabinoids showed pronounced antibacterial activity. The use of analytic methods also demonstrated reproducible cannabinoid extraction.

Effects of extraction methods on protein properties obtained from paddy rice and germinated paddy rice.

Rice protein has attracted considerable attention recently due to its physiological effects. This study extracted the proteins from paddy rice (PR) and germinated paddy rice (GPR) using three methods i.e., alkaline, sodium dodecyl sulfate (SDS) reagent and enzymatic extractions. The extracted proteins or protein fractions were assessed for their properties using various techniques. Data were analyzed by 2'3 factorial design experiment. It was found that germination and extraction methods significantly affected the concentration of protein fractions when analyzed by Bradford assay. Average protein fraction concentration of the GPR was lower than that of PR. SDS-PAGE patterns of protein fractions obtained from PR and GPR using any extraction method displayed similar protein profiles. Three major protein bands at about 13 kDa (prolamin), 22-23 kDa (basic glutelin) and 37-39 kDa (acidic glutelin) with small amount of 57 kDa proglutelin were observed. For amino acid profile, germination increased the content of most amino acids, resulting in the higher content of amino acids in GPR, excepted for some amino acids. When processed with in vitro digestion, protein fractions from GPR exhibited a higher level of digestibility than those from PR as evidenced by the less intensity of the protein bands obtained from SDS-PAGE. Alkaline and SDS reagent extractions provided more digestible protein fractions than enzymatic extraction. Extraction methods also influenced phase transition of protein fractions as investigated by a DSC. Alkaline extraction resulted in protein fractions with higher phase transition temperature than the other methods. For antioxidant capacity, extraction methods as well as germination significantly affected antioxidant capacity of the protein fractions. Enzymatic extraction provided protein fractions with the best antioxidant capacity.

Phytochemical Analysis and Evaluation of Antioxidant and Biological Activities of Extracts from Three Clauseneae Plants in Northern Thailand.

This study established the DNA barcoding sequences (matK and rbcL) of three plant species identified in the tribe Clauseneae, namely Clausena excavata, C. harmandiana and Murraya koenigii. The total phenolic and total flavonoid contents, together with the biological activities of the derived essential oils and methanol extracts, were also investigated. Herein, the success of obtaining sequences of these plant using two different barcode genes matK and rbcL were 62.5% and 100%, respectively. Both regions were discriminated by around 700 base pairs and these had resemblance with those of the Clausenae materials earlier deposited in Genbank at a 99-100% degree of identity. Additionally, the use of matK DNA sequences could positively confirm the identity as monophyletic. The highest total phenolic and total flavonoid content values (p < 0.05) were observed in the methanol extract of M. koenigii at 43.50 mg GAE/g extract and 66.13 mg QE/g extract, respectively. Furthermore, anethole was detected as the dominant compound in C. excavata (86.72%) and C. harmandiana (46.09%). Moreover, anethole (26.02%) and caryophyllene (21.15%) were identified as the major phytochemical compounds of M. koenigii. In terms of the biological properties, the M. koenigii methanol extract was found to display the greatest amount of antioxidant activity (DPPH; IC50 95.54 µg/mL, ABTS value 118.12 mg GAE/g extract, FRAP value 48.15 mg GAE/g extract), and also revealed the highest α-glucosidase and antihypertensive inhibitory activities with percent inhibition values of 84.55 and 84.95. Notably, no adverse effects on human peripheral blood mononuclear cells were observed with regard to all of the plant extracts. Furthermore, M. koenigii methanol extract exhibited promise against human lung cancer cells almost at 80% after 24 h and 90% over 48 h.

The Synthesis of Molecularly Imprinted Polymers on Microcentrifuge Tube Filters for Solid-Phase Extraction and the HPLC-UV Determination of Andrographolides.

Molecular imprinting-based solid-phase extraction has been in the spotlight to improve recognition selectivity and detection sensitivity of andrographolides. The synthesis of molecularly imprinted polymers on micro centrifuge tube filters for the extraction and the determination of andrographolides were investigated. Molecularly imprinted polymers were synthesized using the photo-polymerization method for the preconcentration of andrographolides (AD) template molecule using 2,2-dimethoxy-2-phenylacetophenone as initiators, the mixture of 1-dodecanol and toluene solvent, 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate as functional monomers and cross-linked, respectively. The resultant AD molecularly imprinted polymers (AD-MIPs) were characterized using the Fourier-transform infrared spectrum and scanning electron microscopy. The maximum adsorption of AD-MIPs toward the andrographolides was found to be 85%, and could reach binding equilibrium within 60 min. The sample solution was separated by AD-MIP using solid-phase extraction (SPE). Subsequently, the sample solution was analyzed by the high-performance liquid chromatography (HPLC) method. The AD-MIP could be successfully applied to specifically separate and determine the andrographolides from pharmaceutical products and biological fluid samples with relatively high recoveries (102.01-108.61%). The present method is simple, selective, accurate, and provides a promising alternative to traditional SPE sorbents for the extraction and determination of andrographolides in real samples and biological fluid samples.

Description of four new species of the genus Ptilocerembia Friederichs, 1923 (Embioptera: Ptilocerembiidae) from Thailand.

Four new species of webspinners in the genus Ptilocerembia Friederichs (Ptilocerembiidae) are described including Ptilocerembia thaidina sp. n., P. senathami sp. n., P. catherinae sp. n. and P.  rossi sp. n. . from Thailand. Illustrations of heads, genitalia and papilla of adult males, sternite pattern of adult females together with photographs of adult males, females and their galleries for each species are provided. Notes on field observations and egg mass structure are given for P. catherinae sp. n. A distribution map and a dichotomous key to the Thai species in the genus Ptilocerembia are also included. 

Dachtylembia, a new genus in the family Teratembiidae (Embioptera) from Thailand.

Dachtylembia gen. nov. (Embioptera: Teratembiidae), is described and illustrated based on specimens of a new species (D. siamensis) collected from Thailand. The geographical distribution of this species in Thailand is mapped.

Human-landing patterns of Anopheles dirus sensu lato (Diptera: Culicidae) in experimental huts treated with DDT or deltamethrin.

Anopheles dirus females landing on humans inside experimental huts treated with residual applications of DDT or deltamethrin were observed during the wet season in Pu Teuy Village, Kanchanaburi Province, western Thailand. Two identical experimental huts were constructed in the fashion of typical local rural Thai homes. Pretreatment (baseline) human-landing collections (HLC) in both huts showed an early evening peak of activity between 1900 and 2000 h with no significant difference in numbers of mosquitoes captured between huts over a period of 30 collection nights. During posttreatment HLC, female mosquitoes continued to show greater landing activity inside huts fitted with insecticide-treated panels during the first half of the evening compared with the second half. A greater number (proportion) of An. dirus females landed on humans in the hut treated with deltamethrin compared with DDT. Comparing pre- and posttreatment HLC, the DDT-treated hut showed a 79.4% decline in attempted blood feeding, whereas exposure to deltamethrin resulted in a 56.3% human-landing reduction. An odds ratio was performed to demonstrate the relative probability (risk) of mosquitoes entering and attempting to blood feed in the two treated huts compared with untreated control huts. Mosquitoes were approximately 0.47 times less likely to land on humans inside a DDT-treated hut compared with the deltamethrin-treated hut. Although both chemicals exerted strong excitatory responses, DDT appears to have a more pronounced and significant (P = 0.002) effect on behavior than deltamethrin, resulting in greater movement away from the insecticide source and thus potential reduction of blood-feeding activity.

Genetic structure and gene flow of Anopheles minimus and Anopheles harrisoni in Kanchanaburi Province, Thailand.

Isozyme frequencies were compared in seven field collections of Anopheles minimus complex using starch gel electrophoresis. Mosquito collections were sampled from four districts in Kanchanaburi Province where malaria is endemic. From eight enzyme systems, nine loci and seven polymorphisms were detected, indicating limited genetic differentiation among the seven collections (F(ST) = 0.061). The highest percent polymorphic loci were observed in Bong Ti Noi (BTN) Village (55.6%), whereas the least percent polymorphism was seen in Tha Kradan (TK) Village (22.2%). Comparing villages Pra Jedee (PJ) with Pu Teuy C (PTC) and Huai Khayeng (HK) with Pra Jedee (PJ), gene flow among collections varied from 3.72 to 62.25 reproductive migrants per generation. Among the seven collections, no correlation was seen between genetic and geographical distances (P > 0.05). Anopheles minimus (former species A) and Anopheles harrisoni (former species C) from Pu Teuy fit most closely in the same cluster, possibly indicating relatively recent divergence between taxa. The genetic and epidemiological ramifications of these findings are discussed.

Effects of physiological conditioning on behavioral avoidance by using a single age group of Aedes aegypti exposed to deltamethrin and DDT.

The behavioral and physiological responses of 6-d-old Aedes aegypti (L.) adult females exposed to deltamethrin and DDT were characterized using a free-choice excito-repellency test system. Excluding varying pretest age and carbohydrate availability as possible confounders, insecticide contact (measuring irritancy) and noncontact (measuring repellency) behavioral assays were conducted on two nonbloodfed groups, either unmated or mated (nulliparous), and two blood-fed groups, either parous or newly full-engorged mosquitoes. The degree of escape response to deltamethrin and DDT varied according to the physiological conditioning. Escape rates from contact and noncontact chambers with deltamethrin were more conspicuous in nonbloodfed groups compared with mosquitoes previously bloodfed. There were no significant differences in escape responses between unmated and nulliparous test populations. With DDT, a more pronounced escape response was observed in unmated compared with other physiological conditions. More moderate escape response was seen in nulliparous mosquitoes, and the least was observed in full bloodfed test individuals, regardless of test compound. Ae. aegypti, regardless of pretest conditioning, was completely susceptible to deltamethrin, whereas showing high resistance to DDT. Despite profound differences in resistance, there was no significant difference in avoidance response between chemicals and mosquito conditioning. Moreover, pre- and postbloodmeals were found to influence assay outcome and thus to have relevance on the interpretation of susceptibility and excito-repellency assays.

Biting patterns of Anopheles minimus complex (Diptera: Culicidae) in experimental huts treated with DDT and deltamethrin.

Biting patterns of natural populations of Anopheles minimus s.l. females entering experimental huts treated with DDT and deltamethrin were carried out at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, western Thailand. Two experimental huts, control and treatment, were constructed in the fashion of local Thai homes. Pre-spray biting activity of An. minimus females peaked at 19:00-22:00. Post-treatment exposure continued to show greater landing activity during the first half of the evening. An overall greater proportion of An. minimus females entered the hut treated with deltamethrin compared to DDT. The hut fitted with DDT-treated net panels showed a 71.5% decline in attempted blood feeding, whereas exposure to deltamethrin-treated panels resulted in a 42.8% human-landing reduction. DDT exhibited significantly more pronounced (P < 0.05) effects in overall reduction of biting activity than did deltamethrin.