4th-stage Larvae Research Articles

Determination of microsporidia infection in larvae and adult anopheles mosquitoes (Diptera: Culicidae) Northwest Ethiopia

Microsporidia are a diverse group of obligate intracellular, spore-forming parasites that infect invertebrates including mosquitoes. A few studies showed that microsporidia infection of mosquitoes is associated with larval mortality or reduced adult fecundity and lifespan. This phenomenon might be used to protect against vector-borne disease transmission. This study aimed to determine microsporidia infection in field-collected Anopheles larvae and reared adults. Anopheles larvae were collected from the field and analyzed from February to April 2022. After determining the species identity of the 4th stage larvae, they were dissected to extract their midgut while the early-stage larvae were transported to an entomological laboratory for rearing the adult in the insectary room. Similarly, after determining the species identity of adult female Anopheles mosquitoes, they were dissected to extract their midgut. Then, two thin smears for each dissected larvae and/ adults were made. Fixed preparations were stained with Giemsa and modified Ziehl-Neelsen staining techniques to detect the microsporidia. Descriptive statistics and an independent t-test were used to analyze the data. Anopheles gambiae complex was identified as the most predominant species from field-collected larvae and insectary reared adults. From the total 258 larvae and 258 adults An.gambiae complexes tissue smears examined, microsporidia were detected in 2.7% [7/258, (95%, CI: 0.8-5)] and 1.2% [3/258, (95%, CI: 0.01-2.7)] of larvae and adults respectively. The mean density of microsporidia in larvae and adults was 64.9 (± 23.4 SD) and 36 (± 8.5SD) respectively. The difference in microsporidia density between infected larvae and adults was statistically significant (F=1.77, P= 0.02). Conclusion: Significant level of microsporidia infection was detected using light microscopy. Further microsporidia identification in the genus and species level is needed.

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

BackgroundThere 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).MethodsThe 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.ResultsThe 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 <n->, and n-Hexadecanoic acid bioactive components. These chemical constituents were used as additive formulations in pesticides, pest control, insect repellent, and insecticidal agents.ConclusionsOur 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.

Anguillicola (Anguillicoides) crassus: Morphometric characteristics and pathogenicity in eels (Anguilla anguilla) in Greece.

Anguillicola (Anguillicoides) crassus is a swimbladder nematode parasite of Anguilla anguilla (Linnaeus, 1758). The present study investigates the morphology and pathogenicity of A. crassus in European eel, as well as, the effects of different aquatic environments on the infection of A. crassus, in Greece. A total of 268 nematodes were collected from four estuarine systems in Greece. In all collected parasites, measurements carried out to define the morphological characteristics of parasites: parasite length and width, oesophagus max length and max width. Τhe mean parasite length was 23.50mm (95% CI: 22.42-24.58) for females, 12.95mm (95% CI: 12.25-13.64) for the males and 6.39mm (95% CI: 5.27-7.50) for the juveniles. The mean parasite width was 1.99mm (95% CI: 1.88-2.10) for females, 0.93mm (95% CI: 0.86-1.00) for males and 0.51mm (95% CI: 0.39-0.64) for juveniles. In total, the mean intensity was found 3.15 (95% CI: 2.53-3.78), while the highest mean intensity per eel was 8.00 (95% CI: 0.00-29.51) in Tholi Lagoon and the lowest was 2.20 (95% CI: 0.36-4.04) in Amvrakikos Gulf. Longitudinal and transverse histological sections of two adults A. crassus and two swimbladders were carried out. Multiple granules were observed, as a tissue reaction of the organism to swath around the 4th stage larvae (L4) that have entered the swimbladder. Molecular analysis was performed on three female adults A. crassus derived from eel specimens coming from the Vistonis estuarine system. The high reproductive capacity of the parasite reveals that A. anguilla has low effective defense mechanisms against the parasite. Also, the morphometric variations of the A. crassus seems to have a plastic feature which is being differently expressed when exposed to various environments.

Larvicidal efficacy of some natural products against Culex pipiens larvae

Mosquitoes control, especially through the use of natural products, seems to be an urgent priority. In the current study, efficacy of some of these natural products such as plants extracts, organic acids, and plant oils, in monitoring to check their larvicidal effect, were evaluated by their application on 4th stage larvae of Culex pipiens. For plant methanol extracts; ginger, followed by garlic, showed the highest effect while onion showed the lowest efficacy. The lowest effect was found for peppermint in the oils used the highest for clove oil. Whereas, organic acids have a great effect on 4th stage larvae; citric acid has a higher effect at the concentration of 1.2 ml/100ml than acetic acid. Used materials are related to concentration and time. This research shows promising results in the use of natural products as larvicidal agents for mosquitoes, and further studies are required to examine others

Residual Activity of Pyriproxyfen Against Mosquitoes in Catch Basins in Northwestern Riverside County, Southern California.

A field study was carried out on the year-long residual activity of the insect growth regulator (IGR) pyriproxyfen (Nylar 0.5G) in comparison with methoprene (Altosid® XRP Pellets) against mosquito developmental stages in catch basins in northwestern Riverside County, southern California. Pyriproxyfen was applied at 75, 100, 125, 150, 175 g per catch basin and methoprene at 3.5 g per catch basin. A total of 80 catch basins (10 per each treatment and 20 for control) were used. Posttreatment observations of catch basins were carried out at weekly intervals, with all pupal collections reared to adults. Mosquito species composition in this study, consisting mostly of Culex species (693), was predominated by Cx. quinquefasciatus (92.8%), followed by Cx. erythrothorax (5.5%), Cx. tarsalis (1.2%), Cx. stigmatosoma (0.3%), and Cx. thriambus (0.2%). Activity of both IGRs was expressed as percent inhibition of adult emergence (% IAE). Data generated on % IAE showed that, like methoprene, pyriproxyfen provided complete control of mosquitoes at 75, 125, and 175 g per catch basin up to 50 wk posttreatment at the Riverside amusement park, whereas its activity against mosquitoes in catch basins treated with 100 g and 150 g at the Eastvale site was short-lived, up to 48 wk. Water samples, bioassayed against laboratory-reared, 4th-stage larvae of Cx. quinquefasciatus 1-2 wk after the 50-wk-long study, showed evidence of significant % IAE (∼50) by pyriproxyfen at the 2 higher rates (125 g, 175 g) used at the amusement park. In conclusion, pyriproxyfen can be used to effectively control mosquitoes in catch basins for 48-50 wk, depending on the rate of application.

Conspecific and allospecific larval extracts entice mosquitoes to lay eggs and may be used in attract-and-kill control strategy

One of the strategies of integrated vector management is to lure gravid mosquitoes for surveillance purposes or to entice them to lay eggs in water containing toxins that kill the offspring (attract-and-kill or trap-and-kill). Typically, the major challenge of this approach is the development of a lure that stimulates oviposition plus a toxin with no deterrent effect. Bacillus thuringiensis var. israelensis (Bti) satisfies the latter criterion, but lures for these autocidal gravid traps are sorely needed. We observed that gravid Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus laid significantly more eggs in cups with extracts from 4th-stage larvae (4 L) of the same or different species. No activity was found when 4 L were extracted with hexane, diethyl ether, methanol, or butanol, but activity was observed with dimethyl sulfoxide extracts. Larval extracts contained both oviposition stimulant(s)/attractant(s) and deterrent(s), which partitioned in the water and hexane phases, respectively. Lyophilized larval extracts were active after a month, but activity was reduced by keeping the sample at 4 °C. In the tested range of 0.1 to 1 larvae-equivalent per milliliter, oviposition activity increased in a dose-dependent manner. In field experiments, Ae. aegpti laid significantly more eggs in traps loaded with larval extracts plus Bti than in control traps with water plus Bti.

Larvicidal Properties of Citrus hystrix DC Fruit (Taw-Shaut-Khar-Thee) Extracts against Aedes aegypti Larvae Collected from Different Townships of Yangon

The present study aimed to evaluate the larvicidal and ovicidal activities of ethanol extracts of dried fruits, internal fruit materials and peels of Citrus hystrix DC against Aedes aegtpti larvae from Dagon (North) and Hlinethaya Townships in Yangon Region from May 20l5 to December 2016. The 3rd and 4th stage larvae were exposed for 24 hours to various concentrations of ethanol extracts of different parts of the Citrus hystrix fruit. The dry fruit extract resulted in significantly higher mortality of 100% and 96.4% in Aedes larvae collected from Dagon (North) and Hlinethaya, respectively (P&lt;0.05). The mortality of Aedes larvae in peel extract was found to be 92.5% in Hlinethaya and 94.4% in Dagon (North), respectively. The extract of Citrus hystrix DC fruit was very effective to kill Aedes larvae collected from Dagon (North). When compared with each other, slightly higher mortality of larvae from Dagon (North) was observed at 0.l5 g/100 m1 concentration of all extracts than that of larvae from Hlinethaya. Ovicidal test found 100% protection to lay their eggs at 0.0l25 g/l00 ml dose and persistency test found 100% mortality of 3rd and 4th instar Aedes larvae up to 6 days at a dose of 0.2 g/100 ml fruit extract. The LC50 and LC90 values of 0.0343 g, 0.0415 g, 0.0231 g, and 0.1256 g, 0.1584 g, 0.0742 g for peel, internal material and fruit extracts, respectively, on Aedes larvae from Dagon were found to be lower than 0.0396 g, 0.0552 g, 0.0280 g and 0.1408 g, 0.2322 g, 0.1060 g, respectively, from Hlinethaya. The findings of the present study revealed that the ethanol extract of the fruit of Citrus hystrix DC has strong larvicidal, ovicidal and long-term persistency activity on Aedes mosquitoes as a good source of preparations for mosquito control. The study may contribute to a reduction in the application of synthetic insecticides, which in turnincreases the opportunity for natural control of various medically important insects by botanical insecticides.

EXPERIMENTAL LIFE CYCLE OF CONTRACAECUM QUADRIPAPILLATUM N. SP. IN WHITE PELICAN (PELECANUS ERYTHRORHYNCHUS) AT LAKE NASSER, EGYPT: MORPHOLOGICAL AND GENETIC EVIDENCES

During November 2013 to October 2015, one hundred and nine fish specimens of Clarias lazera were captured from different areas of Lake Nasser, South Egypt. They were found infected with 3rd stage larvae of Contracaecum with 100% prevalence. 4th stage larvae and adults were experimentally obtained from the lower part of esophagus of white Pelicans Pelecanus erythrorhynchus previously administered 3rd stage larvae. Detailed morphological description of both larvae (L3) and adults, by light and scanning electron microscopes as well as molecular analysis of the internal transcribed spacers of nuclear ribosomal DNA (ITS-1 & ITS-2) indicated the presence of a new Contracaecum sp. here named C. quadripapillatum n. sp.

Bifidobacterium animalis subspecies lactis modulates the local immune response and glucose uptake in the small intestine of juvenile pigs infected with the parasitic nematode Ascaris suum

ABSTRACTAn evaluation of a localized intestinal allergic type-2 response concomitant with consumption of probiotic bacteria is not well documented. This study investigated the effect of feeding probiotic Bifidobacterium animalis subspecies lactis (Bb12) or a placebo in weaned pigs that were also inoculated with Ascaris suum (A. suum) eggs to induce a strong Th2-dependent allergic type 2 immune response. Sections of jejunal mucosa were mounted in Ussing chambers to determine changes in permeability and glucose absorption, intestine and liver samples were collected for analysis of type-2 related gene expression, jejunum examined histologically, and sera and intestinal fluid were assayed for parasite antigen specific antibody. The prototypical parasite-induced secretory response to histamine and reduced absorption of glucose in the jejunum were attenuated by feeding Bb12 without a change in mucosal resistance. Parasite antigen-specific IgA response in the serum and IgG1 and IgG2 response in the ileal fluid were significantly increased in A. suum-infected pigs treated with Bb12 compared to infected pigs given the placebo. Ascaris suum-induced eosinophilia in the small intestinal mucosa was inhibited by Bb12 treatment without affecting the normal expulsion of A. suum 4th stage larvae (L4) or the morphometry of the intestine. Expression of genes associated with Th1/Th2 cells, Treg cells, mast cells, and physiological function in the intestine were modulated in A. suum infected-pigs treated with Bb12. These results suggested that Bb12 can alter local immune responses and improve intestinal function during a nematode infection by reducing components of a strong allergenic type-2 response in the pig without compromising normal parasite expulsion.

English

The Culicidae are biting insects that are most harmful to people. They are almost all blood-suckers, and are responsible for the spread of many important diseases such as malaria, yellow fever and elephantiasis. Entomopathogenic microorganisms occupy an important place among the alternative methods of fighting against pests insect. The fungus, Beauveria bassiana is an entomopathogenic agent naturally present in the ecosystems and offers a very interesting potential for controlling populations of mosquitoes. Our study aims at showing the 4th stage larvae histological changes of the domestic mosquito Culex pipiens, treated with the entomopathogenic fungus B. bassiana in suspension which corresponds to a dose of 107 spore/mL. In fact, after developing the individuals in the fungal solution, histological study was carried out on the following parts; cuticle, intestine, adipose tissue and hemolymph. The histological studies showed many alterations and malformation in the treated 4th instar larvae body and tissues. The infection affected the different parts of the body, especially the cuticle, the adipose cells, and midgut, in addition to the development and the colonization of B. bassiana inside tissues. The application of B. bassiana on the cuticle of the larvae was dependent on an apparent disturbance in the structure of the cuticle or the degeneration of its different parts. The fungus infection does not stop at the level of the body walls, so it even goes to adipose tissue, epidermal cells and intestine. Based on these results, B. bassiana could be suggested as suitable biocontrol agent against C. pipiens. Key words: Beauveria bassiana, Culex pipiens, cuticle, histological study, midgut, adipose tissue.

Novel synthesis of gold nanoparticles using Artemisia vulgaris L. leaf extract and their efficacy of larvicidal activity against dengue fever vector Aedes aegypti L.

The Aedes aegypti L. mosquito transmits dengue and yellow fever, which cause millions of death every year. Dengue is a mosquito-borne viral disease that has rapidly spread worldwide particularly in countries with tropical and subtropical climates areas. The present study denotes a simple and eco-friendly biosynthesis of gold nanoparticles using Artemisia vulgaris L. leaf extract as reducing agent. The synthesized gold nanoparticles were characterized by UV-Visible Spectroscopy, X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), Dynamic Light Scattering (DLS), Zeta Potential (ZP), Transmission Electron Microscopy (TEM) and Energy Dispersive X-ray Spectroscopy (EDX). Solid state 13C NMR was utilized to confirm the presence of larvicidal compound Beta caryophyllene in the synthesized AuNPs. Larvicidal activity of the synthesized AuNPs was measured against A. aegypti over 12 and 24h exposure periods and compared with essential oil in various concentrations (25ppm, 50ppm, 100ppm, 200ppm and 400ppm). After a 12h exposure period, the larvicidal activity of 3rd instar larva by AuNPs showed LC50=156.55ppm and LC90=2506.21ppm, while and essential oil displayed LC50=128.99ppm and LC90=1477.08ppm. Larvicidal activity of 4th instar larva by AuNPs showed LC50=97.90ppm and LC90=1677.36ppm, while essential oil displayed LC50=136.15ppm and LC90=2223.55ppm. After a 24h of exposure period, larvicidal activity of 3rd instar larva by AuNPs showed LC50=62.47ppm and LC90=430.16ppm and essential oil showed LC50=111.15ppm and LC90=1441.51ppm. The larvicidal activity of 4th instar larva and AuNPs displayed LC50=43.01ppm and LC90=376.70ppm and for essential oil LC50=74.42ppm, LC90=858.36ppm. Histopathology of A. aegypti with AuNPs for 3rdand 4th stage larvae after 24h exposure at the highest mortality concentration (400ppm) showed that the area of the midgut, epithelial cells and cortex were highly affected. The present findings demonstrate that the biosynthesis of AuNPs using A. vulgaris leaf extracts could be an eco-friendly, safer nanobiopesticide and treatment against A. aegypti which could be used to combat of dengue fever.

Life cycle and morphology of development stages of Physocephalus dromedarii (Nematoda: Spirocercidae)

ObjectiveTo study the development of Physocephalus dromedarii (P. dromedarii) in the final host. MethodsFor this, 5 adult dromedaries were orally infected with third larval stages of P. dromedarii obtained from naturally infected scarab beetles (Scarabaeus cristatus). The camels were necropsied 14, 42, 70, 84 and 280 days after infection and their abomasi were examined for the presence of nematodes. ResultsEarly 4th stage larva occurred already 2 weeks after infection. They were still in the sheet of the 3rd stage larva. Six weeks after infection, the nematodes became juvenile male and female adults measuring 9 and 10 mm, respectively. Their size doubled at 10 weeks post infection and patency was reached at 12 weeks. P. dromedarii was still present in the camel that was examined 40 weeks after infection. ConclusionsAs a result of experimental infection of the natural host, the determined prepatent period of P. dromedarii equalled 12 weeks.

Evaluation of Endod (Phytolacca dodecandra: Phytolaccaceae) as a Larvicide Against Anopheles arabiensis, the Principal Vector of Malaria in Ethiopia.

Malaria control methods rely mostly on adult mosquito control using insecticide-treated nets and indoor residual spraying with insecticides. Plants such as endod (Phytolacca dodecandra) can potentially be used for the control of mosquito larvae as a supplement to adult control methods. Following the discovery of endod, a molluscicide plant, more than 5 decades ago in Ethiopia, subsequent studies have shown that its potency can further be increased by simple procedures such as aging endod berry powder in water. This study was conducted to evaluate the killing effect of fresh and aged endod solution against 4th-stage larvae of Anopheles arabiensis. Laboratory-reared An. arabiensis larvae exposed to different concentrations of endod preparation using distilled or spring water had 50% lethal concentration (LC(50)) = 49.6 ppm and 90% lethal concentration (LC(90)) = 234 ppm for fresh and LC(50) = 36.4 ppm and LC(90) = 115.7 ppm for the aged endod solution in distilled water against the laboratory population. Against field-collected larvae of the same species, aged preparations in habitat water resulted in higher LC(50) (472.7 ppm) and LC(90) (691 ppm) values, with only a slight improvement over fresh preparations in habitat water (LC(50) = 456.2 ppm; LC(90) = 896.1 ppm). In general, although aged preparations of endod required lower concentrations than fresh to kill at least 90% of the larvae, these concentrations were much higher (12-70×) than that required for schistosome-transmitting snails.

Larvicidal activities of chinaberry, neem and Bacillus thuringiensis israelensis (Bti) to an insecticide resistant population of Anopheles arabiensis from Tolay, Southwest Ethiopia

ObjectiveTo elucidate the larvicidal potency of neem, chinaberry and Bacillus thuringiensis israelensis (Bti) to larvae of Anopheles arabiensis under semi-field condition and adult susceptibility/resistance to the conventionally used insecticides in Tolay, Southwestern Ethiopia. MethodsWild collected 3rd and 4th stage larvae were exposed to neem, and chinaberry seed powder dissolved in water and Bti in artificial containers at three treatment levels: 0.2, 0.1 and 0.05 g/m2 and controls were free of treatments. Larval and pupal mortalities were monitored daily and residual activities were determined. The experiments were replicated three times. The World Health Organization tube test for all classes of insecticides was conducted on adult Anopheles arabiensis reared from field collected larvae and pupae. Data were analyzed using STATA software version 11. ResultsIn the first application, neem powder caused 88.9%, 87.9% and 79.4% larval and pupal mortality at 0.2, 0.1 and 0.05 g/m2 after 4.3, 6.0 and 5.7 days, respectively. The corresponding killing effect of chinaberry was 80.3%, 62.1% and 30.3% after 7.0, 7.7 and 8.3 days respectively. Bti at all treatments killed 100% after 24 h except 2.7 days for 0.05 g/m2. Adult mosquitoes were susceptible only for fenitrothion and pirimiphos-methyl with 100% mortality while resistant to deltamethrin, alpha-cypermethrin, etofenprox and dichloro-diphenyl-tricgloroethane with only 9.0%, 3.0%, 5.1% and 2.0% mortalities respectively. ConclusionsNeem, chinaberry and Bti showed potent larvicidal and pupicidal activities. However, in the area, high level of mosquito resistance to pyrethroids and dichloro-diphenyl-tricgloroethane was seen which will pose serious challenge to vector control in the future. Therefore, using integrated approach including these botanical larvicides is warranted to manage insecticide resistance.

Evaluation of Mosquito Repellent Activity of Isolated Oleic Acid, Eicosyl Ester from Thalictrum javanicum.

To evaluate the traditional use, the mosquito repellent property of Thalictrum javanicum and to confirm the predicted larvicidal activity of the isolated compound, oleic acid, eicosyl ester from its aerial parts by PASS software, the present study was carried out using 4th instar stage larvae of the mosquitoes, Aedes aegypti (dengue vector) and Culex quinquefasciatus (filarial vector). Insecticidal susceptibility tests were conducted and the mortality rate was observed after 24 h exposure. The chitinase activity of isolated compound was assessed by using purified β-N-acetyl glucosaminidase (chitinase). Ecdysone 20-monooxygenase assay (radioimmuno assay) was made using the same larval stage of A. aegyptiand C. quinquefasciatus. The results were compared with the crude methanol extract of the whole plant. The isolated compound, oleic acid, eicosyl ester was found to be the most effective larvicide against A. aegypti (LC50/24 h -8.51 ppm) and C. quinquefasciatus (LC50/24 h - 12.5 ppm) than the crude methanol extract (LC50/24 h - 257.03 ppm and LC50/24 h - 281.83 ppm, respectively). The impact of oleic acid, eicosyl ester on reducing the activity of chitinase and ecdysone 20-monooxygenase was most prominent in both the target species, A. aegyptiand C. quinquefasciatus than the control. The results therefore suggest that the compound, oleic acid, eicosyl ester from Thalictrum javanicum may be considered as a potent source of mosquito larvicidal property.

Operational Evaluation Of Vectomax® WSP (Bacillus thuringiensis Subsp. israelensis+Bacillus sphaericus) Against Larval Culex pipiens in Septic Tanks (1).

The residual effectiveness of VectoMax® WSP (a water-soluble pouch formulation containing a combination of Bacillus thuringiensis subsp. israelensis strain AM65-52 and B. sphaericus strain ABTS 1743) when applied to septic tanks against 3rd- and 4th-stage larvae of Culex pipiens L. was evaluated in this study. This formulation was evaluated at operational application rates of 1 pouch (10 g) and 2 pouches (20 g) per septic tank. Both application rates resulted in >96% control of larvae for 24 days. Operationally, VectoMax WSP has proven to be a useful tool for the nonchemical control of Culex species in septic tank environments.

어류 기생성 선충 Anisakis simplex sensu stricto와 Anisakis pegreffii 유충의 excretory-secretory products 및 somatic extracts의 가수분해효소 활성 비교

Department of Marine Bioscience, Gangneung-Wonju National University, Gangneung, 210-702, KoreaHydrolase activities of excretory-secretory products (ESP) and somatic extracts (SE) from Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii larvae were investigated by using API ZYM kit. In esterase group, acid phosphatase showed high activity from both of A. simplex (s.s.) and A. pegreffii. Esterase (C4) showed activity only from SE and A. simplex (s.s.) showed higher activity than A. pegreffii. Alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase showed higher activity in 3rd stage larvae than in 4th stage larvae of both species. In aminopeptidase group, only leucine arylamidase showed remarkable activity in SE of both anisakid species, and A. simplex (s.s.) SE showed higher activity than A. pegreffii SE. In glycosidase group, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase showed higher activity in A. simplex (s.s.) than A. pegreffii, and 4th larvae showed higher activity than 3rd larvae. These differences in hydrolase activity of anisakid nematodes larvae are thought to be due to different metabolism such as growth, moulting, digestion and feeding. Key words: Anisakis simplex sensu stricto, Anisakis pegreffii, Hydrolase activities, API ZYM

English

Volatile oils of Microlobius foetidus were used for the evaluation of mortality, trypsin, chymotrypsin and acetylcholine inhibition and Aedes aegypti morphology. Bioassays were made with different concentrations (25, 50 and 100 &mu;g/ml) and alterations in the gut of 4th stage larvae were observed. Volatile oils affected larvae in all stages, with 100% mortality for the 100 &mu;g/ml concentration (LD50 &gt;33.02). The acute toxic unit (2.7 &mu;g/ml), chronic toxic unit (32.68 &mu;g/ml) and toxic load (2.7 &mu;g/ml) confirm the sensibility of the 4th stage larvae. These larvae and 3rd presented a lesser trypsin (0.176 &mu;mol/min), chymotrypsin (0.110 &mu;mol/min) and acetylcholinesterase (0.172 &mu;mol/min) synthesis. Larvae of the 4th stage also had their internal morphology observed, and the main alterations were discontinuity of the peritrophic epithelium, thickening of the peritrophic membrane, decrease/increase of the subjacent epithelium and decrease of endoperitrophic space were observed in the mid gut. The results show the repellent activity of the volatile oils of M. foetidus, with a retardation of overall growth, that are associated with the inhibition of the trypsin and chymotrypsin synthesis. Mortality and enzymatic inhibition in all developmental stages confirm the insecticide potential of M. foetidus. Key words: Dengue fever, digestive system, biological control agents, natural products.&nbsp;

Secretion of Protective Antigens by Tissue-Stage Nematode Larvae Revealed by Proteomic Analysis and Vaccination-Induced Sterile Immunity

Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H. polygyrus proteins closely implicated in immune modulation and protective immunity.

Laboratory evaluation of Dalbergia oliveri (Fabaceae: Fabales) extracts and isolated isoflavonoids on Aedes aegypti (Diptera: Culicidae) mosquitoes

We explored the potential of Burma rosewood, Dalbergia oliveri in dengue fever vector control by evaluating various extracts as larvicides and growth disruptors of Aedes aegypti Linnaeus (Diptera: Culicidae) under laboratory conditions. Third instars and pupae of the mosquito were tested using WHO guidelines. Highest larvicidal effect was obtained with dichloromethane and hexane extracts (LC50=289.1 and 325.3ppm, respectively) within 24h post-treatment. However, toxicity was about 1.78-fold more in dichloromethane extracts than hexane extract, if treatment was given for longer duration of 48h (LC90=5048.5ppm for hexane and 2848.1ppm for dichloromethane extracts, respectively). In these treatments moderate inhibition of pupation was also seen with some larvae staying as 4th stage larvae for 8 days before dying. There were no significant differences in the sex ratio of emerged adults compared with the controls. Dichloromethane extract, being most active, was subjected to sequential chemical separation to obtain series of isoflavonoids. Three compounds (+)-medicarpin, formononetin and (±)-violanone were identified as active larvicides of Ae. aegypti. Significant pupal mortality was observed with dichloromethane extract treatment of 1–3h old pupae after 48h post-treatment (LC50=1004.5ppm). Isoflavonoid medicarpin (LC50=296.72ppm) was most active compound against pupae. Eclosion inhibition (EI) was also observed in pupae treated with dichloromethane extract (EI50 of 668.7ppm). Thus, naturally occurring larvicides can be particularly effective where larval habitats are readily identifiable. Results obtained should be of value in the search for new natural larvicidal compounds from other extracts as well and represent the first report of this plant species being used to control a mosquito vector.