First Molecular Identification of Caligus clemensi on Cultured Crimson Snapper Lutjanus erythropterus on Jerejak Island, Penang, Peninsular Malaysia.

Fish parasites such as Caligus clemensi are a serious concern for cultured fish in many regions of the world, including Malaysia. This study was designed to elucidate the parasites' prevalence and intensity coupled with the morphology and molecular identification of C. clemensi on cultured Lutjanus erythropterus in Jerejak Island, Penang, Peninsular Malaysia. The study was carried out on 200 fish specimens of cultured L. erythropterus obtained from the GST group aquaculture farm. Parasites were collected from the infested part of L. erythropterus fish, and their prevalence and intensity were determined. The parasites were identified morphologically using a field emission scanning electron microscope. Molecular studies were performed through PCR amplification and sequencing. MEGA 5 was used to construct a phylogenetic tree using the pairwise distance method. The results showed that only the C. clemensi parasite was found prevalent on L. erythropterus fish with a prevalence and mean intensity (S.D) of 198 (99%) and 36.4 ± 12.2, respectively. The prevalence varied significantly with respect to fish length (p < 0.05). The nucleotide BLAST sequence for 18S ribosomal RNA partial sequences showed 97% with 100% query similarity, E-value 0 with C. clemensi with the accession number DQ123833.1. Conclusively, C. clemensi remains a major parasite of L. erythropterus in the study area.

Analysis of chemical compositions and larvicidal activity of nut extracts from Areca catechu Linn against Aedes (Diptera: Culicidae).

BACKGROUND: There is a growing need to use green alternative larvicidal control for Aedes larvae compared to chemical insecticides. Substantial reliance on chemical insecticides caused insecticide resistance in mosquito populations. Thus, research for alternate chemical compounds from natural products is necessary to control Aedes larvae. This study explores the analysis of chemical compositions from Areca catechu nut as a potential larvicide for Aedes (Diptera: Culicidae). METHODS: The Areca catechu nut collected from Ipoh, Perak, Malaysia was grounded into powder and used for Soxhlet extraction. The chemical analysis of the extracts and their structures were identified using the GCMS-QP2010 Ultra (Shimadzu) system. National Institute of Standards and Technology (NIST) Chemistry WebBook, Standard Reference Database 69 (https://webbook.nist.gov/chemistry/) and PubChem (https://pubchem.ncbi.nlm.nih.gov/), the two databases used to retrieve the synonyms, molecular formula, molecular weight, and 2-dimensional (2D) structure of chemical compounds. Next, following WHO procedures for larval bioassays, the extracts were used to asses larvicidal activity against early 4th instar larvae of Aedes aegypti and Aedes albopictus. RESULTS: The larvicidal activities were observed against early 4th stage larvae with different concentrations in the range from 200 mg/L to 1600 mg/L. The LC50 and LC95 of Aedes aegypti were 621 mg/L and 2264 mg/L respectively; whereas the LC50 and LC95 of Aedes albopictus were 636 mg/L and 2268 mg/L respectively. Mortality was not observed in the non-target organism test. The analysis using gas chromatography and mass spectrometer recovered several chemical compounds such as Arecaidine, Dodecanoic acid, Methyl tetradecanoate, Tetradecanoic acid , and n-Hexadecanoic acid bioactive components. These chemical constituents were used as additive formulations in pesticides, pest control, insect repellent, and insecticidal agents. CONCLUSIONS: Our study showed significant outcomes from the extract of Areca catechu nut and it deserves further investigation in relation to chemical components and larvicidal actions between different species of Aedes mosquitoes. Even though all these findings are fundamental, it may have some interesting potentials to be developed as natural bio-larvicidal products.

Systematic Performance Comparison of Fe3+/Fe0/Peroxymonosulfate and Fe3+/Fe0/Peroxydisulfate Systems for Organics Removal.

Activated zero-valent iron (Ac-ZVI) coupled with Fe3+ was employed to activate peroxymonosulfate (PMS) and peroxydisulfate (PDS) for acid orange 7 (AO7) removal. Fe3+ was used to promote Fe2+ liberation from Ac-ZVI as an active species for reactive oxygen species (ROS) generation. The factors affecting AO7 degradation, namely, the Ac-ZVI:Fe3+ ratio, PMS/PDS dosage, and pH, were compared. In both PMS and PDS systems, the AO7 degradation rate increased gradually with increasing Fe3+ concentration at fixed Ac-ZVI loading due to the Fe3+-promoted liberation of Fe2+ from Ac-ZVI. The AO7 degradation rate increased with increasing PMS/PDS dosage due to the greater amount of ROS generated. The degradation rate in the PDS system decreased while the degradation rate in the PMS system increased with increasing pH due to the difference in the PDS and PMS activation mechanisms. On the basis of the radical scavenging study, sulfate radical was identified as the dominant ROS in both systems. The physicochemical properties of pristine and used Ac-ZVI were characterized, indicating that the used Ac-ZVI had an increased BET specific surface area due to the formation of Fe2O3 nanoparticles during PMS/PDS activation. Nevertheless, both systems displayed good reusability and stability for at least three cycles, indicating that the systems are promising for pollutant removal.

The potential use of Azolla pinnata as an alternative bio-insecticide.

Four different tests showed the effectiveness of Azolla pinnata plant extracts against Aedes aegypti and Aedes albopictus mosquitoes. In the adulticidal test, there was a significant increase in mortality as test concentration increases and A. pinnata extracts showed LC50 and LC95 values of 2572.45 and 6100.74 ppm, respectively, against Ae. aegypti and LC50 and LC95 values of 2329.34 and 5315.86 ppm, respectively, against Ae. albopictus. The ovicidal test showed 100% eggs mortality for both species tested for all the concentrations tested at 1500 ppm, 1000 ppm, 500 ppm, 250 ppm and 125 ppm. Both tested samples of Ae. aegypti and Ae. albopictus did not lay any eggs in the plastic cups filled with the A. pinnata extract but instead opted to lay eggs in the plastic cups filled with water during the oviposition deterrence test. Similarly, the non-choice test of Ae. aegypti mosquitoes laid eggs on the sucrose solution meant for the nutrient source of the mosquitoes instead of in the plastic cup that was designed to facilitate oviposition filled with the extract. This clearly indicates the presence of bioactive compounds which are responsible in adulticidal and ovicidal activity in Aedes mosquitoes and at the same time inducing repellence towards the mosquitoes. The LC-MS results showed mainly three important chemical compounds from A. pinnata extracts such as 1-(O-alpha-D-glucopyranosyl)-(1,3R,25R)-hexacosanetriol, Pyridate and Nicotinamide N-oxide. All these chemicals have been used for various applications such as both emulsion and non-emulsion type of cosmetics, against mosquito vector such as Culex pipens and Anopheles spp. Finally, the overall view of these chemical components from A. pinnata extracts has shown the potential for developing natural product against dengue vectors.

Chemical composition and larvicidal activities of Azolla pinnata extracts against Aedes (Diptera:Culicidae).

BACKGROUND: The resistance problem of dengue vectors to different classes of insecticides that are used for public health has raised concerns about vector control programmes. Hence, the discovery of alternative compounds that would enhance existing tools is important for overcoming the resistance problem of using insecticides in vectors and ensuring a chemical-free environment. The larvicidal effects of Azolla pinnata extracts by using two different extraction methods with methanol solvent against Aedes in early 4th instar larvae was conducted. METHODS: The fresh Azolla pinnata plant from Kuala Krai, Kelantan, Malaysia was used for crude extraction using Soxhlet and maceration methods. Then, the chemical composition of extracts and its structure were identified using GCMS-QP2010 Ultra (Shimadzu). Next, following the WHO procedures for larval bioassays, the extracts were used to evaluate the early 4th instar larvae of Aedes mosquito vectors. RESULTS: The larvicidal activity of Azolla pinnata plant extracts evidently affected the early 4th instar larvae of Aedes aegypti mosquito vectors. The Soxhlet extraction method had the highest larvicidal effect against Ae. aegypti early 4th instar larvae, with LC50 and LC95 values of 1093 and 1343 mg/L, respectively. Meanwhile, the maceration extraction compounds were recorded with the LC50 and LC95 values of 1280 and 1520 mg/L, respectively. The larvae bioassay test for Ae. albopictus showed closely similar values in its Soxhlet extraction, with LC50 and LC95 values of 1035 and 1524 mg/L, compared with the maceration extraction LC50 and LC95 values of 1037 and 1579 mg/L, respectively. The non-target organism test on guppy fish, Poecilia reticulata, showed no mortalities and posed no toxic effects. The chemical composition of the Azolla pinnata plant extract has been found and characterized as having 18 active compounds for the Soxhlet method and 15 active compounds for the maceration method. CONCLUSIONS: Our findings showed that the crude extract of A. pinnata bioactive molecules are effective and have the potential to be developed as biolarvicides for Aedes mosquito vector control. This study recommends future research on the use of active ingredients isolated from A. pinnata extracts and their evaluation against larvicidal activity of Aedes in small-scale field trials for environmentally safe botanical insecticide invention.

Larvicidal Effectiveness of Azolla pinnata against Aedes aegypti (Diptera: Culicidae) with Its Effects on Larval Morphology and Visualization of Behavioural Response.

Dengue is vector-borne diseases with 390 million infections per year extending over 120 countries of the world. Aedes aegypti (L.) (Diptera: Culicidae) is a primary vector for dengue viral infections for humans. Current focus on application of natural product against mosquito vectors has been the main priority for research due to its eco-safety. The extensive use of chemical insecticides has led to severe health problems, environmental pollution, toxic hazards to human and nontarget species, and development of insecticide resistance on mosquitoes. Azolla pinnata is an aquatic fern and predominantly used as feed in poultry industry and as fertilizer in agricultural field for enhancing the fertility of rice paddy soil. The present study was conducted to explore the larvicidal efficacy of A. pinnata using fresh and powdered form against late third-stage larvae (6 days, 5 mm in larvae body length) of Ae. aegypti (L.) (Diptera: Culicidae). The larvicidal bioassays were performed using World Health Organization standard larval susceptibility test method for different concentration for powdered and fresh A. pinnata. Powdered A. pinnata concentration used during larvicidal bioassay ranges from 500ppm to 2000ppm; meanwhile, fresh A. pinnata ranges from 500ppm to 9,000,000 ppm. The highest mortality was at 1853 ppm for powdered A. pinnata compared with fresh A. pinnata at 2,521,535 ppm, while the LC50 for both powdered and fresh A. pinnata recorded at 1262 ppm and 1853 ppm, respectively. Finally, the analysis of variance (ANOVA) showed significant difference on Ae. aegypti larval mortality (F=30.439, df=1, p≤0.001) and concentration (F=20.002, df=1, p≤0.001) compared to powdered and fresh A. pinnata at 24-hour bioassay test. In conclusion, the powdered A. pinnata serves as a good larvicidal agent against Ae. aegypti (L.) (Diptera: Culicidae) and this study provided information on the lethal concentration that may have potential for a more eco-friendly Aedes mosquito control program.

Neobenedenia melleni Parasite of Red Snapper, Lutjanus erythropterus, with Regression Statistical Analysis between Fish Length, Temperature, and Parasitic Intensity in Infected Fish, Cultured at Jerejak Island, Penang, Malaysia.

The fish parasites collected from Lutjanus erythropterus fish species showed a correlation with parasitic intensity, fish size, and temperature, and statistical model summary was produced using SPSS version 20, statistical software. Statistical model summary concluded that among the variables which significantly predict the prevalence of Neobenedenia melleni parasites are fish length and water temperature, both significant at 1% and 5%. Furthermore, the increase in one unit of fish length, holding other variables constant, increases the prevalence of parasite by approximately 1 (0.7≈1) unit. Also, increasing the temperature from 32°C to 33°C will positively increase the number of parasites by approximately 0.32 units, holding other variables constant. The model can be summarized as estimated number of Neobenedenia melleni parasites = 8.2 + 0.7 ⁎ (fish length) + 0.32 ⁎ (water temperature). Next, this study has also shown the DNA sequence and parasitic morphology of Neobenedenia melleni. Nucleotide sequence for 18s ribosomal gene RNA in this study showed 99% similarity with N. melleni EU707804.1 from GenBank. Finally, all the sequence of Neobenedenia melleni in this study was deposited in GenBank with accession numbers of KU843501, KU843502, KU843503, and KU843504.