Surachai Pikulkaew

The knockdown of the maternal estrogen receptor 2a (esr2a) mRNA affects embryo transcript contents and larval development in zebrafish.

In zebrafish, ovulated oocytes are loaded with maternal estrogen receptor 2a (esr2a) mRNA which is spread as granular and filamentous structures throughout the central ooplasm and is promptly relocated inside the blastodisc area at the 1-cell stage (0.2h post-fertilization, hpf), as shown by in situ hybridization. This transcript is available for translation until its sharp decline from 4 to 8 hpf, being replaced by low levels of zygotic esr2a mRNA mainly localized in the head region and around the yolk sac from 24 hpf until hatching at 48 hpf. To test the functional role of the maternal esr2a mRNA, 1- or 2-cell embryos were injected with 10.3 ng each of morpholino (MO) to knockdown translation (MO2-esr2a) of both maternal and zygotic esr2a transcripts, with a missplicing MO (MO3-esr2a) to effectively block post-transcriptionally the zygotic transcript alone, and with a non-specific MO-control. Treatment with MO2-esr2a increased apoptosis in embryos, especially in the brain, and caused severe malformations in 63% of 1-5 dpf larvae, as compared to 10-11% in those treated with MO3-esr2a and MO-control. Defects included body growth delay with curved shape, persistent yolk sac with reduced sub-intestinal veins and swollen yolk extension, abnormal brain and splanchnocranium development, smaller eyes and otic vesicles, pericardial oedema, uninflated swim bladder and rudimentary caudal fin with aberrant circular swimming. Affected larvae could survive for only 12-14 days. The MO2-esr2a phenotype was rescued with co-injection of 30 pg/embryo of mutated zebrafish esr2a mRNA encoding the full length of Esr2a, but containing eight silent mutations in the region recognised by MO2-esr2a. A lower dosage (15 pg) failed to recover mortality and abnormality. Raising the dosage to 60 and 90 pg increased abnormality, but not mortality, whereas with 120 pg both mortality and abnormality worsened, indicating a strict quantitative requirement of Esr2a. Co-injection of an anti-p53 MO failed to rescue the MO2-esr2a phenotype, eliminating the possibility of off-target effects. Pangenomic microarray analysis revealed that 240 and 219 significantly expressed transcripts were up- and down-regulated, respectively, by maternal Esr2a protein deficiency in 8-hpf MO2-esr2a embryos. Also at 48 hpf, 162 and 120 presumably zygotic transcripts were up- and down-regulated, respectively, but only 18 were in common with each of the 8-hpf sets. In total, the transcripts from 705 genes were affected by Esr2a knockdown. These findings suggest the involvement of maternal esr2a mRNA, presumably transactivated by maternal 17β-estradiol stored in the oocyte from enveloping granulosa cells, in the epigenetic programming of zebrafish development.

Effects of Alpinia galanga oil on anesthesia and stress reduction in Oreochromis niloticus

Oreochromis niloticus (Nile tilapia) is one widely cultured fish in Thailand. Handling processes and transportation causes high stress in Nile tilapia. This study explores anesthetic effect and stress reduction of Alpinia galanga oil (AGO) on Nile tilapia. The anesthetic activity was evaluated by the time for fish induction to anesthesia and full recovery. It was found that the suitable dose of AGO that caused desirable anesthesia of Nile tilapia was 700 mg/L. This dose gave induction and recovery times of approximately 257 and 438 sec, respectively. Blood glucose and plasma cortisol of the fish anesthetized with AGO showed nearly normal levels indicating that the fish stress during handling was not increased. Study on loading densities of fish mimicked general fish transportation and showed that loading density of fish was a crucial factor on fish stress. The highest water quality was found in the lowest loading density of fish. Water containing AGO at a concentration of 150 mg/L showed significantly higher potential for reducing fish activity and water improvement than without AGO. Therefore, AGO is a promising natural edible plant oil for anesthesia in Nile tilapia.

Development of chrysin loaded poloxamer micelles and toxicity evaluation in fish embryos

Poloxamer micelles promise safety and efficacy for many water insoluble drugs. Chrysin has been reported to have anticancer, anti-inflammatory, antioxidant, and anti-aromatase activities but its water insoluble properties limit its pharmaceutical application. In the present study, chrysin loaded poloxamer micelles were developed. Two types of poloxamers, Pluronic F-68 and Pluronic F-127 were compared. It was found that chrysin loaded Pluronic F-68 micelles (CS-P68) and chrysin loaded Pluronic F-127 micelles (CS-P127) obviously increase the aqueous solubility of chrysin. The results also indicated that the type of polymer and ratio of drug to polymer affected size and desirable characteristics of the micelles. The micelle system of CS-P68 and CS-P127 formed at drug to polymer ratios of 1:4 and 1:2, respectively, was found to be the most suitable monodispersed system with a nanosize-range diameter. The in vivo study in zebrafish eggs indicates that the toxicity of CS-P68 and CS-P127 is a dose response. CS-P68 and CS-P127 at a drug dose of 10 ng/mL or less is safe for zebrafish embryo growth. The results of this study indicate enhanced water solubility of chrysin. Chrysin loaded poloxamer micelles are promising for further use in in vivo studies in mammalian animals and humans.

Self-microemulsifying drug delivery system and nanoemulsion for enhancing aqueous miscibility of Alpinia galanga oil

Alpinia galanga oil (AGO) possesses various activities but low aqueous solubility limits its application particularly in aquatic animals. AGO has powerful activity on fish anesthesia. Ethanol used for enhancing water miscible of AGO always shows severe side effects on fish. The present study explores the development of self-microemulsifying drug delivery systems (SMEDDS) and nanoemulsions (NE) to deliver AGO for fish anesthesia with less or no alcohol. Pseudoternary phase diagrams were constructed to identify the best SMEDDS-AGO formulation, whereas NE-AGO were developed by means of high-energy emulsification. The mean droplet size of the best SMEDDS-AGO was 82 ± 0.5 nm whereas that of NE-AGO was 48 ± 1.6 nm. The anesthetic effect of the developed SMEDDS-AGO and NE-AGO in koi (Cyprinus carpio) was evaluated and compared with AGO ethanolic solution (EtOH-AGO). It was found that the time of induction the fish to reach the surgical stage of anesthesia was dose dependent. NE-AGO showed significantly higher activity than SMEDDS-AGO and EtOH-AGO, respectively. EtOH-AGO caused unwanted hyperactivity in the fish. This side effect did not occur in the fish anesthetized with SMEDDS-AGO and NE-AGO. In conclusion, SMEDDS and NE are promising delivery systems for AGO.

Molecular epidemiology and geographical distribution of Nosema ceranae in honeybees, Northern Thailand

Abstract Objective To determine the contamination levels of Nosema ceranae in honeybees and its molecular linkages in different geographical areas of Northern Thailand. Methods Seventy-eight apiaries in Northern Thailand were chosen at random. The detection was accomplished both by microscopic examination and multiplex PCR. Nosema positive samples were evaluated by PCR sequencing and phylogenetic analysis. Results Of the samples subjected to microscopic examination, 11.54% were found to be positive for Nosema while 29.49% of the samples evaluated by PCR were found to be positive for the disease. Honeybees from four of the six provinces surveyed in Northern Thailand were positive for Nosema , with the highest prevalence in Chiang Mai Province (48.57%). There was a high diversity of Nosema strains in some locations, while the same strain of pathogen was identified in many locations in Northern Thailand. Conclusions This is the first report about the contamination levels and distribution pattern of nosemosis in Thailand. The study found the same group of Nosema in different locations, and different groups of Nosema in the same location. This pattern of distribution will be an advantage for disease control in the future.

Anesthetic activity of plant essential oils on Cyprinus carpio (koi carp)

The aims of this study were to investigate the anesthetic and cytotoxic effects of essential oils (EOs) of Ocimum basilicum (OBO), O. canum (OCO), and O. sanctum (OSO) on Cyprinus carpio (koi carp). For anesthetic effect, induction time to surgical anesthesia and recovery time were determined. For cytotoxicity effect, viability of fish peripheral blood nuclear cells (PBMCs) was investigated. Results indicated that increasing oil concentration caused significant (p < 0.01) decrease of induction time. OSO at 100, 200, and 300 mg/L gave the induction time of 169.5 ± 10.2, 62.8 ± 2.3, 45.3 ± 2.2 sec, respectively, significantly shorter than OCO, and OBO. The recovery time of anesthetized fish was dose dependent (p <0.01). Among them, OCO showed the longest recovery time of 313.0 ± 8.1, 420.7 ± 12.6, 616.6 ± 12.1 sec for concentrations of 100, 200, and 300 mg/L, respectively, followed by OSO and OBO, respectively. Within 10 min contact time of the EOs and fish PBMCs, the fish PBMC viability was higher than 80%. Increase contact time and EO concentration caused an increase in cytotoxicity to fish PBMC. OBO showed less toxic than OSO and OCO. Based on the desired induction and recovery times for anesthetizing koi carp, OBO, OCO, and OSO at 300, 200, and 100 mg/L, respectively were suggested to be the most suitable. It was concluded that OBO, OCO, and OSO can be used as natural anesthetics for fish.

Investigation of antiaromatase activity using hepatic microsomes of Nile tilapia (Oreochromis niloticus)

Microsomal aromatase enzymes of humans and rats have been used in antiaromatase assays, but enzyme activity is species-specific. The current study extracted hepatic microsomes of Nile tilapia (Oreochromis niloticus) to investigate and compare the antiaromatase activity of chrysin, quercetin, and quercitrin. This activity was evaluated using a dibenzylfluorescein (DBF) assay. Results revealed that the age and body weight of Nile tilapia affected the yield of extracted microsomes. Extraction of hepatic microsomes of Nile tilapia was most effective when using a reaction medium with a pH of 8.0. A DBF assay using Nile tilapia microsomes revealed significant differences in levels of antiaromatase activity for chrysin, quercetin, and quercitrin. Chrysin was the most potent aromatase inhibitor, with an IC50 of 0.25 mg/mL. In addition, chrysin is an aromatase inhibitor that also inhibits the proliferation of cancer cells. Hepatic microsomes of Nile tilapia can be used to investigate and compare the antiaromatase activity of different compounds.

Influence of clove oil and eugenol on muscle contraction of silkworm (Bombyx mori)

Clove oil is used in fish anesthesia and expected to have a mechanism via glutamic receptor. The present study explores the activities of clove oil and its major compound, eugenol, in comparison with L-glutamic acid on glutamic receptor of silkworm muscle and fish anesthesia. It was found that clove oil and eugenol had similar effects to L-glutamic acid on inhibition of silkworm muscle contraction after treated with D-glutamic acid and kainic acid. Anesthetic activity of the test samples was investigated in goldfish. The results demonstrated that L-glutamic acid at 20 and 40 mM could induce the fish to stage 3 of anesthesia that the fish exhibited total loss of equilibrium and muscle tone, whereas clove oil and eugenol at 60 ppm could induce the fish to stage 4 of anesthesia that the reflex activity of the fish was lost. These results suggest that clove oil and eugenol have similar functional activities and mechanism to L-glutamic acid on muscle contraction and fish anesthesia.

Nanoemulsion: A suitable nanodelivery system of clove oil for anesthetizing Nile tilapia

Clove oil ethanolic solution (CL-EtOH) have always been used for fish anesthesia. However, ethanol causes major side effect of fish hypersensivity. In this study, clove oil loaded nanoemulsion (CLN) was developed in order to enhance water miscibility of clove oil without using ethanol in the preparations. The obtained CLN was characterized in terms of droplet size, size distribution expressed as polydispersity index (PDI), and zeta potential. The anesthetic effect of CLN in comparison with CL-EtOH on Oreochromis niloticus (Nile tilapia) was investigated. The results showed that the best CLN was composed of 20% w/w clove oil and 15% w/w polysorbate 20. This CLN has internal droplet size of 63.2 ± 1.0 nm, PDI of 0.31 ± 0.04, and zeta potential of - 30.3 ± 8.1 mV. GC-MS analysis indicated that eugenol was the main compound in clove oil. It was found that the induction time to anesthesia for Nile tilapia that received this CLN was shorter than that received CL-EtOH at the same eugenol concentration. The results of this study showed the potential of nanoemulsion on water miscible and efficacy enhancing of clove oil without using ethanol. The obtained CLN from this study is a promising formulation for fish aquaculture where fish sedation is required.

Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee

Abstract Objective To develop loop-mediated isothermal amplification (LAMP) to detect Nosema ceranae (N. ceranae) in honeybee samples. Methods LAMP primers were designed recognizing six distinct fragments of 16s rRNA gene and LAMP reaction was determined by optimizing the concentration of reagents, such as forward inner primer and backward inner primer, deoxynucleoside triphosphate and betaine, time and temperature. Ten-fold serial dilutions of DNA were used to determine the detection limit and accuracy using both LAMP and PCR tests. Results LAMP required 1.2 μmol/L of forward inner primer and backward inner primer primers, 0.2 μmol/L of forward outer primers and backward outer primer, 2 μmol/L of Mg2+, 0.6 mol/L of betaine, 0.6 μmol/L of deoxynucleoside triphosphate, 4.8 IU of Bst DNA polymerase and 30 ng of DNA. The optimal temperature was 63 °C and after a 40-min incubation time, a clearly ladder-like pattern of LAMP product appeared in the gel electrophoresis. LAMP appeared more sensitive than a standard PCR in detection of N. ceranae. Conclusions LAMP gave a good results and it could be an alternative diagnostic tool instead of PCR to detect N. ceranae infection in honeybee.