3rd-stage Larvae Research Articles

Rat lungworm (Angiostrongylus cantonensis) active larval emergence from deceased bubble pond snails (Bullastra lessoni) into water.

Angiostrongylus cantonensis (the rat lungworm) is a zoonotic parasite of non-permissive accidental (dogs, humans, horses, marsupials, birds) hosts. The 3rd stage larvae (L3s) in the intermediate host (molluscs) act as the source of infection for accidental hosts through ingestion. Larvae can spontaneously emerge from dead gastropods (slugs and snails) in water, which are experimentally infective to rats. We sought to identify the time when infective A. cantonensis larvae can autonomously leave dead experimentally infected Bullastra lessoni snails. The proportion of A. cantonensis larvae that emerge from crushed and submerged B. lessoni is higher in snails 62 days post-infection (DPI) (30.3%). The total larval burden of snails increases at 91 DPI, indicating that emerged larvae subsequently get recycled by the population. There appears to be a window of opportunity between 1 and 3 months for infective larvae to autonomously escape dead snails. From a human and veterinary medicine viewpoint, the mode of infection needs to be considered; whether that be through ingestion of an infected gastropod, or via drinking water contaminated with escaped larvae.

Seroprevalence of Ascaris suum compared to milk spot prevalence at slaughter in Italian fattening pigs

Ascaris suum is one of the most important parasites of pigs. Apart from liver condemnation due to lesions caused by migrating larvae ("milk spots"), A. suum infections can compromise weight gain, feed conversion efficacy, as well as meat quality. The true prevalence of infection depends on the diagnostic test used and is often underestimated. We compared liver inspection at slaughter with serology, based on the recognition of a purified A. suum haemoglobin or complete homogenate of the 3rd stage larvae isolated from lungs, in nine pig farms in northern Italy. Liver lesions were found on all farms with prevalence ranging from 3.8% to 98.3%. All farms were also positive for circulating antibodies against As-Hb and As-Lung-L3, with prevalence among pigs on each farm ranging from 36.4-100% and 54.5-100%, respectively. Seroprevalence was consistently higher when compared to the prevalence of milk spots at slaughter. The higher sensitivity of the ELISA tests combined with their ease of use makes them an interesting tool to evaluate A. suum infection levels.

Susceptibility to Organophosphates in Culex quinquefasciatus from Colombia.

Susceptibility to organophosphates was evaluated in 2 populations of Culex quinquefasciatus from the department of Atlantico, Colombia. Bioassays for temephos, malathion, and pirimiphos-methyl were performed with 3rd-stage larvae and adult females of Cx. quinquefasciatus from the municipalities of Soledad and Puerto Colombia, following the methods of the World Health Organization and Centers for Disease Control and Prevention, respectively. The median lethal concentration (LC50) and 90% lethal concentration (LC90) resistance ratios (RRLC50 and RRLC90) were determined for each insecticide in the field populations evaluated, using the Cartagena strain as the susceptible control. Relative to LC50 and LC90 of the Cartagena strain, the population from Puerto Colombia was moderately resistant to temephos (RRLC50 5.7-fold) and malathion (RRLC50 8.6-fold, RRLC90 9-fold) and susceptible to pirimiphos-methyl (RRLC50 and RRLC90 < 5-fold). The population from Soledad was susceptible to temephos and pirimiphos-methyl (RRLC50 and RRLC90 < 5-fold) and showed moderate resistance to malathion (RRLC50 7.5-fold). It is important to emphasize that routine monitoring of insecticide resistance in Cx. quinquefasciatus helps us detect resistance early and improve the effectiveness of control strategies.

Evaluation of tea tree oil formulations contact and stomach toxicity against the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval, 1883) (Lepidoptera: Noctuidae)

The insecticidal potential of tea tree oil formulations was tested for contact and stomach poison toxicities against various stages of the Spodoptera littoralis ((Boisduval, 1883) (Lepidoptera: Noctuidae) larvae under laboratory conditions. The study was carried out at Yozgat Bozok University Faculty of Agriculture between 2020 and 2021. In the contact toxicity test, the formulations were tested at different stages of larvae by topical application. Among the tested formulations, TTO (100%), F14 (91.72%), and F15 (89.20%) formulations caused the highest mortality in the S. littoralis 3rd stage larvae after 72 h. Dose-response bioassay with the most promising formulations (TTO, F14 and F15) revealed that LD50 values were 0.016, 0.046, and 0.076 μg/larvae for TTO, F14, and F15, respectively. The stomach poison effects of the formulations were tested by applying a 0.16 μg/cm$^{2}$ dose to lettuce leaf discs. The F17 and F18 formulations produced the highest mortality with mortality rates of 75% and 65% after 10 days of incubation, respectively. The calculated LC$_{50}$ values for these formulations were 0.027 and 0.042 μg/cm$^{2}$ for F17 and F18 formulations after 10 days of incubation, respectively. These results revealed that tea tree oil and its main components containing formulations have the potential in controlling this destructive lepidopteran pest species.

Ancylostoma ceylanicum infections in humans in Vietnam.

Ancylostoma ceylanicum is recognized as the only zoonotic hookworm species that is able to mature into adult stage in the human intestine. While human infections caused by this hookworm species have been reported from neighboring countries and this hookworm is prevalent in dogs in Vietnam, human infection has never been reported in Vietnam. The present study, therefore, aimed to identify human infections with A. ceylanicum in Vietnam. A total of 526 fecal samples from the residents in Long An Province were collected and the presence of hookworm eggs was detected by the Kato-Katz method. The results indicated that the overall prevalence of human hookworm infection was 85/526 (16.2%). After filter paper culture, 3rd stage larvae were successfully obtained from 48 egg-positive samples. The larvae were identified for their species using semi-nested PCR-RLFP on the cox1 gene. As a result, two hookworm species were confirmed; single species infections with Necator americanus or A. ceylanicum, and mixed infections with both species were found in 47.9%, 31.3%, and 20.8% of the samples, respectively.

Detection of Gnathostoma spinigerum Advanced 3rd-Stage Larvae in the Chinese Edible Frog, Hoplobatrachus rugulosus, from Local Markets in Phnom Penh, Cambodia.

The Chinese edible frogs, Hoplobatrachus rugulosus (n=20), and the striped snakehead fish, Channa striata (n=34), were purchased from local markets in 3 administrative regions of Cambodia (Phnom Penh, Pursat, and Takeo Provinces) from May 2017 to April 2019, and their infection status with Gnathostoma sp. larvae was investigated. The frogs and fish were transported to the laboratory with ice and examined using the artificial digestion method. Advanced 3rd-stage larvae (AdL3) of Gnathostoma spinigerum, 24 in total number (1–6 larvae/frog), were detected from 6 (60.0%) out of 10 frogs purchased from Phnom Penh. No gnathostome larvae were detected in 10 frogs purchased from Takeo Province and 34 snakeheads from Phnom Penh, Pursat, and Takeo Provinces. AdL3 isolated from the frogs were 2.55–3.90 mm long and 0.31–0.36 mm wide. They had a characteristic head bulb (0.081×0.191 mm in average size) with 4 rows of hooklets, a muscular long esophagus (0.950–1.230 mm long), and 2 pairs of cervical sacs (0.530–0.890 mm long). The average number of hooklets in the 1st, 2nd, 3rd, and 4th rows was 41, 45, 48, and 51, respectively. These features were consistent with G. spinigerum AdL3. By the present study, it has been first confirmed that the Chinese edible frog, H. rugulosus, from Phnom Penh serves as a second intermediate host for G. spinigerum, although their intensity of infection was not so high compared to other previously reported localities.

Prospecting for special metabolites and larvicidal activity of ethanolic extracts from Azadirachta indica A. Juss. (Neem), collected in Tauá-CE against Aedes aegypti mosquito larvae

In the search for an alternative control against Aedes aegypti, many types of research are developed to discover substitutes for synthetic insecticides, including the use of oils, extracts, or active constituents from plants to find new insecticidal substances. The present work describes the phytochemical study results and evaluation of toxicity against Aedes aegypti larvae of ethanol extracts from Azadirachta indica A. Juss collected in Tauá-CE. Phytochemical tests were performed by different methods involving colorimetric, precipitation reactions, various metabolites such as alkaloids, anthocyanidins, anthocyanins, steroids, and flavonols, flavonones, triterpenoids was evident in the active extracts. For the toxicity tests, different concentrations of ethanolic extracts (250, 500, 1000 and 2000 μg / mL) were prepared with sterile distilled water and dimethylsulfoxide - DMSO (1%), and then 20 mL of each solution was added. And 25 3rd stage larvae in plastic cups. All bioassays were performed in quadruplicate. DMSO in an aqueous solution was used as a negative control. As a result, the tested extracts proved toxic to Aedes aegypti larvae, with an LC50 value higher than the control (10%) and ranging from 12% to 46%. The results obtained show that all extracts have an important pharmacological and toxicological potential. They deserve to be investigated in studies for the isolation and identification of bioactive compounds.

AcanR3990 qPCR: A Novel, Highly Sensitive, Bioinformatically-Informed Assay to Detect Angiostrongylus cantonensis Infections.

Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

Feeding Fungal-Pretreated Corn Straw Improves Health and Meat Quality of Lambs Infected with Gastrointestinal Nematodes.

Simple SummaryNon-chemical strategies to control gastrointestinal nematode (GINs) infections are urgently needed to support the sustainable development of the livestock industry. The potential anti-parasitic properties in fungal-pretreated corn straw on health and meat quality of lambs infected with GINs were investigated in this study. In summary, feeding fungal-pretreated corn straw improved health and meat quality, including meat color and tenderness. Improved meat traits were attributed to fungal-pretreated corn straw providing additional dietary protein for lambs and secreting some nematicidal metabolites to repel GINs, which increased PCV and plasma iron content of lambs and reversed negative effects of GINs on meat quality.Infections with gastrointestinal nematodes (GIN) adversely affect meat color in lambs. Although white-rot fungi (WRF) pretreatment increases nutritional value and fiber digestion of corn straw for lambs, whether it can improve meat quality of lambs infected with GINs is unknown. The objective of this experiment was to study effects of feeding WRF-pretreated corn straw on the health and meat quality of lambs infected with GINs. Sixteen healthy Ujumqin lambs were orally drenched with 3rd-stage GINs larvae and randomly divided into two dietary treatments of control (CON) and WRF diets for 70 days of feeding. Results showed that feeding WRF-pretreated corn straw decreased L* and b* values (p < 0.05) and increased a* value (p < 0.01) of both longissimus thoracis et lumborum (LTL) and semimembranosus (SM) muscles of lambs infected with GINs. Feeding WRF-pretreatment corn straw decreased fecal egg count (p = 0.014) and increased packed cell volume (p = 0.013) of lambs from 28 d of feeding and increased plasma iron content (p = 0.008) of lambs from 56 d of the feeding. Feeding WRF-pretreatment corn straw decreased myosin heavy-chain (MyHC)-I (p = 0.032) and MyHC-IIα (p = 0.025) content in LTL muscle and MyHC-I (p = 0.022) and MyHC-IIβ (p = 0.048) in SM muscle of lambs. In conclusion, although there were no significant changes in the content of most amino acids or increased intensity of better flavor compounds, meat quality and health of lambs infected with GINs was significantly improved by feeding WRF-pretreated corn straw due to increased PCV and meat color and tenderness.

Expression of cytosolic and thiolated proteome of Musca domestica larvae under oxidative challenge.

The study was aimed to investigate the expression of cytosolic and thiolated proteins of Musca domestica larvae under oxidative stress. Proteins from acute treatment of hydrogen peroxide (LC50 = 21.52% (v/v)) on 3rd stage larvae of housefly were extracted and purified using an activated Thiol Sepharose® for thiolated protein purification. Two dimensional gel electrophoresis was used for visualizing and analyzing expression of cytosolic and thiolated proteins. Protein spots with more than 5 fold of expression change were identified using liquid chromatography- tandem mass spectrometry (LC-MS/MS). The cytosolic proteins were actin, tropomyosin, ubiquitin, arginine kinase, pheromone binding protein/general odorant binding protein, and ATP: guanidino phosphotransferase. The thiolated proteins with more than 5 fold change in expression as an effect to the acute treatment were fructose bisphosphate aldolase, short chain dehydrogenase and lactate/malate dehydrogenase. The proteins identified in the study should provide vital information for future reference in oxidative stress defence and response occurring in houseflies.

Comparative micro-morphological and phylogenetic analysis between Rhinoestrus purpureus and Rhinoestrus usbekistanicus (Diptera: Oestridae) larvae and its adults

Rhinoestrus purpureus and Rhinoestrus usbekistanicus (Diptera: Oestridae) cause specific nasal myiases of family equidae and are of importance in the horse medicine since it causes severe respiratory diseases. During an epidemiological survey in donkeys from Beni Suef government, Egypt; some morphological and taxonomical doubts arose concerning the identification of Rhinoestrus spp. 1st, 2nd and 3rd stage larvae based on the features of the posterior spiracles and the distribution of dorsal spines on the third segment. Four different morphotypes were retrieved: R. usbekistanicus, R. purpureus and two morphotypes with shared features. The genes encoding for the mitochondrial cytochrome oxidase I (COI) and for the ribosomal subunits 16S and 28S of the four morphotypes of Rhinoestrus were investigated to determine whether they belonged to a single taxon or they displayed genetic differences indicative of more than one species. The genes showed a very low level of sequence variation (COI 0–0.43%, 16S 0–1.45%, 28S 0–0.23%) falling within the intraspecific ranges. Finally, the peritreme features and the spinulation of the third segment of the two morphotypes examined could not be used to differentiate the Rhinoestrus spp.

Immunolocalization of Disorganized Muscle Protein-1 in Different Life Stages of Human Lymphatic Filariid, Brugia malayi.

PurposeWe recently identified disorganized muscle protein-1 of Brugia malayi (DIM-1bm) as a vaccine candidate for human lymphatic filariasis. The present study was aimed at investigating the localization of DIM-1bm in the life-stages of B. malayi to identify the tissue target of vaccine action.MethodsRecombinant DIM-1bm (rDIM-1bm) was prepared and antibodies were raised in BALB/c mice. Immunoblots of SDS-PAGE resolved B. malayi infective 3rd stage larvae (L3) and adult worm antigens and rDIM-1bm were prepared and reacted with anti-rDIM-1bm sera. Sections of adult female worms and whole-mount preparations of L3 and microfilariae (mf) were stained by immunofluorescence using rDIM-1bm antibodies and Alexa Fluor 488 labeled secondary antibodies, and examined under a confocal microscope.ResultsImmunofluorescence staining showed that DIM-1bm is localized mainly in the subcuticular muscle layer in the L3 and the adult worms; no fluorescent signal could be detected in mf.ConclusionThe localization of DIM-1bm in the parasites’ muscle layer suggests that the immunoprophylactic efficacy of DIM-1 is evidently due to immobilization of the parasite and its subsequent immune elimination.

Differential survival of 3rd stage larvae of Contracaecum rudolphii type B infecting common bream (Abramis brama) and common carp (Cyprinus carpio)

The main fish host reaction to an infection with third stage anisakid nematode larvae is a response in which host immune cells (macrophages, granulocytes, lymphocytes) in affected internal organs initially are attracted to the parasite whereafter fibroblasts may enclose the parasite forming granuloma. Generally, the reaction is non-lethal to the parasite which may survive for years in the fish host retaining infectivity to the final host. This may also apply for the anisakid nematode Contracaecum rudolphii (having the adult stage in cormorants, using copepods as first intermediate/paratenic host and zooplankton feeding fish as paratenic hosts). The present study has shown that most Contracaecum rudolphii larvae survive in bream (Abramis brama) (from Lake Balaton, Hungary) whereas the majority of the nematode larvae die in Cyprinus carpio (from Lake Hévíz, directly connected to Lake Balaton). Both cyprinid host species interacted with the nematode larvae through establishing a marked cellular encapsulation around them but with different effects. The differential survival in common carp and bream may theoretically be explained by ecological factors, such as the environmental temperature which either directly or indirectly affect the development of nematode larvae, and/or intrinsic host factors, such as differential immune responses and host genetics.

The Effect of Niclosamide on Certain Biological and Biochemical Properties of Drosophila melanogaster

ABSTRACT Objective: Laboratory research on management strategies such as biological and chemical control usually demands rearing high numbers of high-quality insects. For this reason, there is a need for detailed information about which antimicrobial agents should be added to their diets in order to obtain high quality insects. Materials and methods: Niclosamide, which is an anthelmintic derivative of salisilanilid, was added in amounts of 100, 200, 400 and 800 mg/L into artificial diets of Drosophila melanogaster (Meigen). The effects on survival rate, developmental time and adult longevity of the insects were examined. Furthermore, the effect of this anthelmintic antibiotic on important oxidative stress indicators; lipid peroxidation product, malondialdehyde (MDA) and protein oxidation products; protein carbonyl (PCO) contents and a detoxification enzyme, glutathione S-transferase (GST) activity in 3rd instar larvae, pupae and adult stage of the insect were also investigated. Results: Compared with those insects on the control diet, the tested concentrations of niclosamide significantly decreased survival rate in all developmental stages of the insect. The control diet produced 94.0 ± 1.0 % of 3rd stage larvae whereas in the highest concentration (800 mg/L) this ratio decreased to 14.00 % ± 1.73. While 42.08 ± 0.50 % of the adults survived in the control diet, longevity decreased to 2.30 ± 0.15 days in the highest concentration. All concentrations of niclosamide resulted in increased MDA contents in the last larval stage of D. melanogaster and concentrations of 100, 200 and 800 mg/L niclosamide increased pupal MDA content in comparison to the control group. At low concentrations of niclosamide, PCO decreased in the last stage of larvae. The diet with 400 mg/L niclosamide concentration significiantly increased GST activity in pupal stage. Conclusion: The results of this work indicated that the negative effects of niclosamide on biological characteristics of D. melanogaster are due to an increase in the oxidative stress and crippled detoxification capacity of the insect.

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.

Anti-Ascaris suum IgG antibodies in fattening pigs with different respiratory conditions

During their migration through the pig’s body, Ascaris suum larvae cause significant damage to the lungs. Little is known about the actual impact of this tissue damage on the occurrence and severity of respiratory problems in industrial pig fattening farms. In this study, we evaluated the link between the serological response to two different A. suum antigen preparations and respiratory or meat inspection outcomes. Two different serological tests were used that measure antibodies against either the A. suum haemoglobin molecule or complete homogenate of the 3rd stage larva that migrate through the lungs. Firstly, serum samples were analysed that were collected from 19 herds in which the cause of acute clinical respiratory symptoms was either Actinobacillus pleuropneumoniae, A. suum, or a miscellaneous cause. This was done to test whether serological results could confirm pathological findings. Secondly, serum samples from 60 herds of finishing pigs with a history of high or low frequency of pleuritis at meat inspection (MI), but without acute respiratory symptoms at the time of sampling, were also submitted for serological evaluation using both tests. Regression models were used to search for potential associations between the proportion of pigs testing seropositive with MI results, in particular pathological changes related to the lungs. The results of both ELISAs were strongly associated (P < 0.001) with pigs belonging to a herd where the respiratory problems could be attributed to A. suum by histology, indicating that both tests can be used to diagnose clinical respiratory outbreaks due to A. suum. In the herds without acute clinical respiratory symptoms, a positive association was found between the proportion of pigs testing seropositive and the percentage of livers rejected due to milk spots and with whole carcass condemnations. No association was found between Ascaris serology and lung pathology (pneumonia and pleuritis) registered at MI, however, challenging the likely involvement of Ascaris in the development of these lesions.

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.

INSECTICIDAL ACTIVITY OF ANACARDIUM HUMILE (ANACARDIACEAE) NUT SHELL LIQUID AGAINST AEDES AEGYPTI (DIPTERA: CULICIDAE)

This work investigated the insecticidal activity of Cashew Nut Shell Liquid (CNSL) extracted from the species Anacardium humile, a native plant in the Cerrado biome, against Aedes aegypti. A. humile fruits were collected and kept in a forced ventilation kiln at 40oC for seven days for CNSL extraction. Mortality tests were performed on third stage larvae, pupae and adults. In addition, female oviposition behavior, egg viability, and the residual effect of the solution on 3rd stage larvae were observed. The CNSL was diluted in decreasing gradient concentrations to obtain the Lethal Concentration (LC). In oviposition LC99 was used for larvae. Residual effect and oviposition tests were performed with LC99, LC 50 and LC90 were respectively obtained, 6.63 and 11.23 ppm for larvae. No mortality was observed in adults. The residual effect of the CNSL was five days in LC99. A repellent effect of CNSL on the females was noted, with a significant reduction of egg numbers and a change in egg stratification patterns in the oviposition substrate. The larval hatching rate of the eggs exposed to the substrate moistened with CNSL was significantly lower when compared to the control. The results evidenced the insecticidal activity of A. humile CNSL, suggesting it as a promising product in the quest for new botanical insecticides.

The prevalence of Gasterophilus intestinalis (Diptera: Oestridae) in donkeys (Equus asinus) in Egypt with special reference to larvicidal effects of neem seed oil extract (Azadirachta indica) on third stage larvae.

Gasterophiline larvae are of veterinary and medical importance caused specific equine intestinal myiasis. Gasterophilus intestinalis (Botfly larvae) had a wide geographical distribution. The present study explores the prevalence rate of G. intestinalis 3rd stage larvae in Egypt from January- December 2017; besides, in vitro trials to control of this larvae and evaluation of this trial using Scanning Electron Microscope (SEM) and histopathology of treated larvae. In the present study, the 3rd larval stage of G. intestinalis was found in clusters in the epithelium of the investigated stomach and infested with prevalence rate 97.2%. The highest collected numbers of larvae were found in two months; March and August (570 & 520 larvae) and lowest numbers (200 larvae) were collected in October, November, and December. The calculated LC50 and LC90 values of neem seed extract were 707.9 ppm and 1090.7 ppm. The different alteration was recorded after exposure to oil extract which showed some destruction on cuticle surface as folded and corrugated cuticle, destruction of maxillae with pits on its surface, disfigure and irregularity of cephalic spines. Histopathology of exposed G. intestinalis larvae showed different changes as thinning of cuticle at the different level (exocuticle, endocuticle, cell layers), degeneration of epithelial cells of the gut and different degree of necrosis was described. Life cycle of G.intestinalis was followed up after treatment with neem seed extract.

Preliminary Results On the Efficacy of Macroalgal Extracts Against Larvae of Aedes albopictus.

Activity of ethanolic extracts of the algae Ulva rigida, Asparagopsis taxiformis, Dictyota dichotoma, and Cystoseira barbata, collected along Italian coasts, was tested against larvae of the Asian tiger mosquito ( Aedes albopictus), a vector of pathogens for animals and humans. Tests were carried out using 10 specimens of 3rd-stage larvae per test, following the World Health Organization standard protocol with minor modifications. Among algal extracts, only D. dichotoma was active against the larvae of Ae. albopictus. Ethanolic extracts of that species showed LC90 (the concentration that kills 90% of larvae) and LC50 (the concentration that kills 50% of larvae) values at 44.32 and 85.92 mg/liter, respectively. Based on the data obtained, D. dichotoma biometabolic extracts could be potential candidates as larvicide compounds to control Ae. albopictus, encouraging the use of macroalgae as natural resources in integrated vector management strategies.