Understanding the interplay between growth and gonadal development is crucial for breeding programs and teleost sexual development. Here the growth and gonadal development of captive-reared false clown anemonefish ( Amphiprion ocellaris ) aged one to eight months was investigated. Histological analysis and quantification of insulin-like growth factor-1 ( igf1 ) mRNA transcripts were performed. The fish displayed a maximum specific growth rate of 0.56 ± 0.11% day − 1 at two months with a negative allometric length-weight relationship. Gonadal differentiation occurred at three months, with concurrent oogenesis and spermatogenesis at four months. Male germ cell development intensified from five to eight months, at the stage of perinucleolus oocytes were observed. Expression levels of igf1 in the liver peaked at eight months; expression was highest in gonadal tissues at two months and decreased significantly in older fish. These findings highlight the potential role of igf1 in ovarian differentiation and the growth of primary oocytes. Further investigations are needed to explore the interplay between igf1 expression and other regulatory factors. This research enhances our understanding of fish reproductive biology and has implications for the captive management of false clownfish.

Normalization of reference genes through the selection of appropriate housekeeping genes is important for the reliable quantification of mRNA analyzed using real-time polymerase chain reaction (qRT-PCR). The suitability of various housekeeping genes (HKGs), were valuated as internal references to verify the levels of the contaminant bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane) by determining the level of gene expression of cytochrome P450 4 (cyp4) within the digestive gland of green mussels (Perna viridis)—a species used for biomonitoring environmental levels. Additionally, the effect was explored of mussel gender and maturity status (juvenile or mature) on the stability of the expression of the reference genes. Three candidate HKGs were examined, namely 18S rRNA, 28S rRNA and β-actin. The expression stability was calculated using three statistical approaches—NormFinder, BestKeeper and a comparative threshold cycle (Ct) method. The study found that of the three candidate reference genes, 28S rRNA and 18S rRNA were the most stable with no significant differences when maturity, gender and exposure to BPA were considered. It was noted that when these genes (18S rRNA, 28S rRNA, β-actin) were used for normalization using animals exposed to 10 ng BPA and the qRT-PCR method, different levels of cyp4 expression were observed. However, BPA significantly decreased the expression levels of cyp4 compared to the control (animals not exposed to BPA), only when 18S rRNA was used as an internal reference. These results highlighted the importance of selecting appropriate reference genes as the endogenous control in qRT-PCR in biomonitoring programs of BPA.

The occurrence of the endocrine disruptors bisphenol-A (BPA) and 17 beta-estradiol (E-2) in cultured populations of green mussel (Perna viridis) and water samples collected from selected fresh and marine coastal environments along the eastern coast of Thailand were investigated. Analysis found high levels of BPA in mussel tissues that correlated with levels found in coastal (maximum 37.13 ng/L) and freshwater (50.7 ng/L) sites situated near industrial and densely populated areas. By comparison, high levels of E-2 (62.99 +/- 5.03 ng/L) were found in freshwater sites near to urban areas. Higher concentrations of BPA and E-2 were found in mature green mussels (> 6 mo. old; 6.40 +/- 0.52 cm shell length) than levels determined in juveniles (< 2-3 mo. old; 2.29 +/- 0.65 cm shell length). To evaluate the potential risks associated with the consumption of green mussels, the bioconcentration factor (BCF) for BPA was determined to be 1650 for adult bivalves and 283 for juveniles. As P. viridis can accumulate BPA from the environment, this raises concerns regarding the risks posed by consuming seafood sourced from zones near to major conurbations. Whether the high levels of E-2 found in green mussels is due to accumulation or to de novo synthesis as seen in other molluscs, requires further investigation. While industrial and domestic wastewater may be important sources of BPA, E-2 contamination within the eastern part of the Gulf of Thailand has been linked to domestic waste. The study highlights the importance of the temporal and spatial monitoring of sentinel species, such green mussels, for environmental contaminants, the results of which can lead to the construction of regional risk maps helping to inform national strategies regarding aquaculture zoning and aquatic food safety.

ทำการคนหาเครองหมายทางชวภาพเพอใชตรวจสอบการปนเปอนของสารในกลม Endocrine Disrupting Chemicals (EDCs) ในสงแวดลอมทางนำดวยเทคนค cDNA-AFLP กบหอยเจดย ( Cerithidea cingulata ) ตวเตมวยทไดรบสาร 17β-estradiol (E 2 ) โดยวธการแชทความเขมขน 100 ng/L เปรยบเทยบกบกลมควบคมทไมไดรบสาร E 2 สามารถคดเลอกแถบดเอนเอ 1 แถบทปรากฏในกลมทไดรบสาร E 2 คอ “217_AFLP” เมอแปลรหสเปนกรดอะมโนแลวเทยบเคยงกบฐานขอมล GenBank ณ เดอนมนาคม 2555 พบวามความเหมอนสงสด (46% Identities; 32/72) กบยน transposase ของป Marbled crab ( Pachygrapsus marmoratus ) (GenBank accession no. CAJ76996) และเมอศกษาระดบการแสดงออกของ “217_AFLP” ในเนอเยอสวนเทาของหอยเจดยตวเตมวยดวยปฎกรยา semi-quantitative PCR ทใชยนควบคมภายใน 28S rRNA พบวาทงหอยเจดยเพศเมยและเพศผทไดรบสาร E 2 ทความเขมขน 10 ng/L เปนเวลานาน 7 วน มการแสดงออกของ “217_AFLP” เทากบ 1.45±0.06 และ 1.27±0.18 ตามลำดบ แตกตางจากกลมควบคม (0.45±0.01 และ 0.61±0.06 ตามลำดบ) อยางมนยสำคญทางสถต (p<0.05) ดงนนเครองหมายดเอนเอ “217_AFLP” สามารถใชตดตามตรวจสอบการปนเปอนของสาร E 2 ในสงแวดลอมทางนำได

Cytochrome P450 1A (CYP450 1A) induction is used widely as a biomarker when assessing exposure to contaminants in environmental systems including pesticides. The effect of chlorpyrifos and carbaryl on CYP450 1A induction was assessed both by measurements of the induction of the EROD activity and the CYP450 1A gene expression in hybrid catfish (Clarius gariepinus x Clarius macrocepharus). Fish were treated with several concentrations of chlorpyrifos (0.43, 4.3 or 43 µM), or carbaryl (1.19, 11.9 or 119 µM) for 24 or 48 h. The livers were then assayed for changes. Chlorpyrifos and carbaryl at all concentrations slightly increased the levels of EROD activity after 24 and 48 h and increased gene expression of CYP450 1A mRNA in dose-dependently. Exposure to chlorpyrifos and carbaryl at high concentrations resulted in significant elevation of CYP450 1A gene expression in comparison to control fish (P < 0.05) at both time intervals. These results showed that the responses of CYP450 1A were more pronounced in gene expression analysis than in EROD assay. This suggests that the induction of CYP450 1A at the gene level was more sensitive than at the protein level. The induction of CYP450 1A following chlorpyrifos and carbaryl exposure indicates the possible use of CYP450 1A as a biomarker for detecting effects of these pesticides in hybrid catfish.

Adult workers of Apis cerana, Apis florea and Apis mellifera from colonies heavily infected with Nosema ceranae were selected for molecular analyses of the parasite. PCR-specific 16S rRNA primers were designed, cloned, sequenced and compared to GenBank entries. The sequenced products corresponded to N. ceranae. We then infected A. cerana with N. ceranae spores isolated from A. florea workers. Newly emerged bees from healthy colonies were fed 10,000, 20,000 and 40,000 spores/bee. There were significant dosage dependent differences in bee infection and survival rates. The ratio of infected cells to non-infected cells increased at 6, 10 and 14 d post infection. In addition, hypopharyngeal glands of bees from the control group had significantly higher protein concentrations than infected groups. Bees infected with 40,000 spores/bee had the lowest protein concentrations. Thus, N. ceranae isolated from A. florea is capable of infecting another bee species, impairing hypopharyngeal gland protein production and reducing bee survival in A. cerana.

After serving as an eurotransmitter, acetylcholine is hydrolyzed by acetylcholinesterase (AChE). Inhibition of AChE is considered to be a specific biomarker for exposure to organophosphate and carbamate insecticides. In this study the AChE gene was isolated from hybrid catfish brain by RT-PCR methods using degenerate primers. The amplified fragment of 966 nucleotides generated by PCR was cloned, and sequence analysis showed 82% nucleotide identity with AChE of the Electrophorus electricus. Specific primers of the hybrid catfish AChE gene were then synthesized and used in the examination of AChE gene expression in brain tissue of hybrid catfish exposed to sublethal concentrations of chlorpyrifos (0.43, 4.3 and 43 microM) and carbaryl (1.19, 11.9 and 119 microM) for 24 hr. Real-time PCR was used to compare with the amplified 28S rRNA gene. AChE gene expression was significantly elevated 12.4 times in catfish exposed to 43 microM chlorpyrifos in comparison to the control group (p<0.05). Carbaryl did not produce any significant change. These results indicate thatAChE is more sensitive to administration of chlorpyrifos than carbaryl. The induction of the AChE gene indicates the possibility of using this gene as biomarker for detecting effects of organophosphate insecticides in hybrid catfish.

A means of discriminating among species of clown anemonefishes, based on restriction enzyme analysis of partial mitochondrial DNA sequences, was investigated. Mitochondrial 16S rRNA and cytochrome b genes from 6 species (7 strains) of anemonefish (Premnas biculeatus, Amphiprion polymnus, A. sandaracinos, A. perideraion, A. ocellaris, A. ocellaris var. and A. percula) were PCR-amplified. A 623-bp portion of 16S rRNA gene was obtained from different fishes using the same pair of primers. Further investigation of this 16S rRNA fragment, by restriction endonuclease digestion with BfuCI and RsaI, was not able to distinguish all fishes studied, but did yield 3 different digestion patterns. The first was specific to P. biculaetus, the sole member of the genus Premnas, while the remaining two separated the Amphiprion species into 2 groups: 1) A. polymnas, A. sandaracinos and A. perideraion, and 2) A. ocellaris, A. ocellaris var. and A. percula. In contrast to this, restriction endonuclease digestion of a 786-bp fragment of the cytochrome b gene with HinfI and RsaI, was able to differentiate different 7 anemonefishes. This utility marker is valuable for unambiguous species/strain identification of juvenile anemonefishes.

Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.

This study focused on sensitivity and detection of Streptococcus agalactiae from tilapia (Oreochromis niloticus) by using polymerase chain reaction (PCR) and culture methods. Genomic DNA, extracted by GF-1 bacterial extraction kit, was used as a template for PCR reaction with 16S rRNA primers from S. agalactiae isolated from moribund tilapia. The results showed a significant rapid detection of S. agalactiae when using 0.1 ng of DNA template in a 14 μl PCR reaction. Multiplex PCR was also developed using a pair of primers from 16S rRNA of S. agalactiae (F1/IMOD) in combination with specific primers (L99/Oce215R) in the region of 28S rRNA S. agalactiae detection in tilapia's (IP injection) blood samples were divided into 5 concentration levels: 0, 10, 10, 10, 10 and 10 CFU/ml (n=5). Within 7 days, application of multiplex-PCR assay for rapid detection of S. agalactiae in the experimental infected tilapia gave positive reaction on the 1 day post-infection in tilapia at the concentration of 10 CFU/ml to 10 CFU/ml (60%). In contrast, bacterial culture gave positive result on the 6 day at the concentration of 10 CFU/ml to 10 CFU/ml (20%). The PCR result show that the PCR technique is an effective tool for rapid detection of S. agalactiae than the traditional technique. Therefore, it could be a useful alternative to the culture base method for a routines diagnosis of streptococcal infection in fish.