Evaluation of the Pesticide Emamectin and Methanol Extract of Wheat Bran against Biomphalaria Alexandrina Snails, Their Hemocytes and Their Infection with Schistosoma Mansoni

The present study was carried out to evaluate the molluscicidal activity of the pesticide Emamectin (5% aqueous solution) and the methanol extract from the wheat bran (MEWB) against B alexandrina and their infection with Schistosoma mansoni was studied. The LC90 and LC50 values for Emamectin were 50.4 and 22.3 ppm, respectively. Infection of snails with S. mansoni under the effect of the tested agents was evaluated via four experimental groups, each of 50 snails For three consecutive days, one group of snails was exposed to 9.08 ppm aqueous Emamectin solution, another group received 100 ppm methanol extract of wheat bran (MEWB), a third group was administered by a combination of 9.08 ppm Emamectin and 100ppm MEWB, The fourth group was control maintained under similar experimental conditions. After three days, all the experimental groups were infected with S. mansonimiracidia and observed till shedding of cercariae. The physiological and histological changes were assessed before and after the infection.The changes in hemocytes of infected snails after administration with LC25 of Emamectin or wheat bran was significantly (20% and 30%, respectively) suppressed compared to 75% for the control snails. However, the snails treated with joint of Emamectin and MEWB were the least infected snails (10%). On the other hand, the biochemical test results showed a remarkable reduction of GOT (p<0.01) GPT (p<0.05) and total protein (0.05) in the heamolyph extracted from the snails treated with an aqueous Emamectin solution. Yet, the levels of GOT were significantly increased in the groups administered with the MEWB alone (p<0.01) or in combination with the Emamectin (p<0.001). As for the total protein levels, there were slightly declining (p<0.05) in the group exposed to the aqueous Emamectin and to the contrary, theses levels were significantly increased (P<0.05) in the snails of the two other experimental groups when compared to the controls. Moreover, the haemocytes cells showed a differentiation which varied in number when detected under the microscope. Administration of the aqueous Emamectin solution resulted in a significant increase (p<0.05) of the amaebocytes and a considerable decrease (p<0.05) in the number of granulocytes and hyalinocytes compared to the control snails. On the contrary, a remarkable surge (P<0.05) of granulocytes and decrease of the amaebocytes was detected in the haemolyph of snails treated with MEWB

GAS CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS AND ANTIOXIDANT ACTIVITY OF PUNICA GRANATUM L. PEELS AND ITS ROLE AS IMMUNOSTIMULANT AGAINST SCHISTOSOMA MANSONI INFECTION IN BIOMPHALARIA ALEXANDRINA

Objective: To evaluate the antioxidant activity and chemical composition of Punica granatum L. and test it as immunostimulants against Schistosoma mansoni infection to Biomphalaria alexandrina snails.Methods: Antioxidant activity was evaluated by measuring the free radical scavenging activity of the 90% defatted methanol extract (90% DM) of P. granatum peels and its sub-derived fractions was evaluated via 2,2'-diphenyl-1-picrylhydrazyl and its chemical constituents were identified via gas chromatography-mass spectrometry (GC-MS) analysis. B. alexandrina snails were exposed to pomegranate extracts (PEs) for 1 month before their challenging with S. mansoni miracidia. Infection rates, immunological and histological parameters were, then, evaluated in PE-exposed snails and compared to controls.Results: The antioxidants activities of PE, expressed as scavenging concentration at 50%, were in the following order; 90% DM (12.45) ˃n-butanol(15.59) ˃ethyl acetate (21.36) ˃water (49.16) µg/ml, compared to 7.50 µg/ml for ascorbic acid. The infection rates of PE-exposed snails were 20%,50%, 60%, 70%, and 80%, respectively, for 90% DM, n-butanol, ethyl acetate, water, and dichloromethane extracts compared to 95% in control snails. The number of amoebocytes showed a significant increase, clear differentiation, and size increment in exposed snails compared to controls. Moreover, hermaphrodite glands histology shows a full maturity in the formation of reproductive cells in PE-exposed snails. The GC-MS analysis of the 90% DM extract revealed the presence of 36 compounds representing 93.1% of the total composition. Piperidin-4-ol, 1,3-dimethyl-2,4,6-triphenyl (19.87%), and 6,11-dihydroxy-5,12 naphthacenequinone-1-carboxylic acid (7.80%) were the major components.Conclusion: The identified compounds in 90% DM extract of P. granatum may be responsible for the high antioxidant activity of the fruit and it may account for its immunostimulatory effect against S. mansoni infection in B. alexandrina.Keywords: Punica granatum L., Antioxidant activity, Biomphalaria alexandrina, Schistosoma mansoni, Infection rate, Gas chromatography-mass spectrometry

Potential Correlation between Carboxylic Acid Metabolites in Biomphalaria alexandrina Snails after Exposure to Schistosoma mansoni Infection

Carboxylic acids play an important role in both aerobic and anaerobic metabolic pathways of both the snail and the parasite. Monitoring the effects of infection by schistosome on Biomphalaria alexandrina carboxylic acids metabolic profiles represents a promising additional source of information about the state of metabolic system. We separated and quantified pyruvic, fumaric, malic, oxalic, and acetic acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to detect correlations between these acids in both hemolymph and digestive gland gonad complex (DGG's) samples in a total of 300 B. alexandrina snails (150 infected and 150 controls) at different stages of infection. The results showed that the majority of metabolite pairs did not show significant correlations. However, some high correlations were found between the studied acids within the control group but not in other groups. More striking was the existence of reversed correlations between the same acids at different stages of infection. Some possible explanations of the underlying mechanisms were discussed. Ultimately, however, further data are required for resolving the responsible regulatory events. These findings highlight the potential of metabolomics as a novel approach for fundamental investigations of host-pathogen interactions as well as disease surveillance and control

Phytochemical analysis of Eucalyptus camaldulensis leaves extracts and testing its antimicrobial and schistosomicidal activities

Abstract Background The emerging evolution of antibiotic and anthelmintic resistance and inefficiency of some synthetic drugs elicit the need to investigate new drug sources. In this context, Eucalyptus camaldulensis is an evergreen tree that has been widely used in traditional medicine for the treatment of various health disorders. Results Organic solvent extracts from Eucalyptus camaldulensis leaves were assessed for their antimicrobial activity. Among these extracts, ethyl acetate (EtOAc) and water extracts showed the highest antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. EtOAc extract was further subjected to vacuum liquid chromatography (VLC) technique for the isolation of its polyphenolic ingredients. VLC yielded 13 fractions (1–13) that were assayed for their antimicrobial activities. Six of these fractions, namely 4, 5, 6, 7, 8, and 9, showed antimicrobial activity against all the tested microbes except the fungus Aspergillus niger. Fractions 5 and 6 having considerably the highest antimicrobial activity with inhibition zones ranged from 5 to 14 mm. Moreover, fraction 5 was tested as a larvicidal agent against miracidia and cercariae of Schistosoma mansoni. At concentration of 200 mg/L, the mortality rates of miracidia and cercariae of Schistosoma mansoni were 30%, 20%, 50%, 40%, 80%, 20%, and 100%, 80% at 5, 10, 15, and 20 min, respectively. Chromatographic isolation of the EtOAc extract led to identification of six compounds: gallic acid (1), taxifolin (2), methyl gallate (3), quercetin (4), luteolin (5), and hesperidin (6). Conclusions Ethyl acetate extract from Eucalyptus camaldulensis leaves showed a potent antimicrobial and antischistosomal activity. This activity may be attributed to the six phenolic compounds identified through structure elucidation. Thus, these compounds can be good candidates for treatment of microbes and for the control of schistosomiasis

Double labeling of contralateral histamine-like immunoreactive neurons projecting toward the cerebral ganglionin <i>B</i>. <i>glabrata</i>.

<p><b>A:</b> Biocytin backfill of the right CBc labeled fibers in the buccal commissure (Bc) and numerous cells on the dorsal surface of the left buccal ganglion (magenta). Dashed box encloses area shown at higher magnification in panel D. <b>B</b>: Many of the histaminergic neurons on the dorsal surface of the left ganglion (green) exhibited similar sizes and locations to those labeled by the backfill. Dashed box encloses area shown at higher magnification in panel E. <b>C:</b> Merging of panels A and B demonstrates that several histaminergic neurons project to the CBc (double labeled neurons appear white). Dashed box encloses area shown at higher magnification in panel F. Calibration bar = 50 μm, applies to panels A-C. <b>D:</b> Higher magnification of central region of the left dorsal buccal ganglion. Image contains neurons that were labeled only by the backfill (diamonds) and cells that also contained histamine immunoreactive material (asterisks). <b>E:</b> Same region of the ganglion contains neurons that were labeled only by the immunohistochemistry protocol (triangles) and others that were double-labeled (asterisks). <b>F:</b> Overlay of panels D and E confirms the presence of backfilled neurons (magenta, diamonds), histaminergic neurons (green, triangles), and double-labeled cells (white, asterisks). Symbols mark representative neurons, but not all cells in each class. Calibration bar = 10 μm applies to panels D-F.</p

Histamine-like immunoreactivity in the buccal ganglia of <i>Biomphalaria</i>.

<p><b>A:</b> Approximately 30 histamine-like immunoreactive neurons were present on the caudal surface of each buccal ganglion of <i>B</i>. <i>glabrata</i>. One population of neurons (stars) lay medially, and generally posterior to the buccal commissure. A single pair of larger (20–30 μm) more intensely labeled cells (arrows) flank the buccal commissure (Bc). A second population (single asterisks) formed an oblique band across each ganglion, while a third cluster (brackets) was located along the lateral margin. A few cells of a group located primarily on the ventral surface of the right ganglion are also indicated (double asterisks). The parabuccal nerves (PBn) and the esophageal trunks (Et) are rich in HA-immunoreactive fibers. Calibration bar = 50 μm. <b>B:</b> Cells comprising a medial cluster (star), central oblique band (single asterisk) and lateral population (bracket) can also be seen on the dorsal surface in a posteriorly rotated left buccal ganglion of <i>B</i>. <i>alexandrina</i>. Calibration = 50 μm. <b>C:</b> An encapsulated cluster (arrow) of immunoreactive neurons adhered to each esophageal trunk, near the ganglion. <b>D:</b> The parabuccal nerves project to the mouth region, where they branch to produce a rich innervation. Immunoreactive fibers also cover the salivary gland (arrow). Calibration bar = 100 μm.</p

HA-like immunoreactivity in the cerebral ganglia.

<p><b>A:</b> Dorsal view of the left cerebral ganglion of <i>B</i>. <i>glabrata</i>. The white arrow and double asterisks denote clusters along the lateral margin of the dorsal surface while the white arrowhead indicates an identified individual neuron at the base of the tentacular nerve (Tn). The star, single asterisk and bracket indicate ventral clusters that are not clearly in focus. <b>B:</b> Higher magnification view of area in part A indicated by the rectangle with the arrow showing an axon projecting from a cell in the lateral cluster. <b>C:</b> A deeper focus showing additional cells along the ventral surface in a similar region of another specimen of <i>B</i>. <i>glabrata</i>. <b>D:</b> Medial and, <b>E</b>: lateral regions of the right cerebral ganglia of <i>B</i>. <i>alexandrina</i>, as seen from a ventral vantage. The confocal z-stack spanned the thickness of the ganglion so that both dorsal and ventral cells are shown in the projection and many of the same cells and cell clusters indicated by stars, asterisks and double asterisks can be recognized here as described above for <i>B</i>. <i>glabrata</i>. One cluster of neurons (D, asterisk) is situated near the origin of the cerebral commissure (Cc) and another (E, star) near the cerebral-pleural connective (CPlc). The anterolateral cluster of neurons observed on the dorsal surface (A, double asterisk) continues to the anterior ventral surface (E, double asterisk) near the origin of the cerebral-buccal connective and lip nerves (out of view). Additional individual cells are located anterolaterally on the dorsal surface (E, large solid white arrows), and near the center of the ganglion on the ventral surface (D, large solid white arrows), and one solitary cell (E, small solid white arrow) at the base of the cerebral-buccal connective (diagonally above and out of view of this image). Numerous immunoreactive fibers can be observed in the cerebral commissures and connectives (large, solid white arrowheads). <b>F:</b> Ventral view of the left cerebral ganglia of <i>B</i>. <i>alexandrina</i>. Two clusters of neurons are observed, one (star) located at the origin of cerebral pleural connective (CPlc) and the other (asterisk) near the cerebral commissure (out of view to the left of this image). The subesophageal cerebral commissure contains a pair of fine immunoreactive filaments exiting the anterior ventral surface of the cerebral ganglia (contrasted black arrow). Scale bars = 50 μm, all panels.</p

HA-Like immunoreactivity in the left parietal and visceral ganglia of <i>Biomphalaria alexandrina</i>.

<p><b>A:</b> Left parietal ganglion contains a single large, intensely immunoreactive neuron (large solid white arrow) on the dorsal surface near the center of the ganglion with a thick axon projecting into the underlying neuropil (small solid white arrowhead). This cell near two pairs of lightly stained, large neurons (brackets). A large dim oval cell (inside rectangle) is located anterior to the parietal-visceral connective (LPaVc) projecting an axon toward the visceral ganglion (insert). A cluster of large, lightly stained cells (asterisk) can be seen on the posterior and lateral edge of the dorsal surface. Numerous fibers run in the parietal-pleural connective (PaPlc) including some prominent, thick axons (contrasted black arrow). <b>B:</b> Visceral ganglion. A prominent intense immunoreactive, spherical, bipolar neuron (rectangle) with two main axonal projections (right insert) giving rise to small numerous sub-branches. At different focal plane, another intense monopolar cell can be noticed superficial to the previously described neuron, with axonal projection toward the parietal-visceral connective (left insert). Note along the posterior lateral edge of the visceral ganglion situated dorsally a group of small separated intense neurons (small, solid white arrowheads) in addition to a large heavily stained neuron (arrow) with noticeable axon projecting toward the ganglionic body (large, solid white arrowhead). <b>C:</b> Ventral surface of the visceral ganglion (Vg). Histamine-like immunoreactive material is present in two clusters of 4–5 large neurons, including a group of cells (30–40 μm diameter, arrow) at the anterolateral margin of the ganglion near the right parietal-visceral connective (RPaVc) and a second medial group (25–30 μm diameter, arrowhead) near the left parietal-visceral connective (LPaVc). <b>D:</b> Higher magnification of medial histminergic cluster on the ventral surface of the visceral ganglion. Stout axons (arrows) project from these cells to the LPaVc. The fibers system connecting the left parietal and visceral ganglia together through the connective and connects the two ganglia with their follower ones (<b>A, B</b> contrasted black arrowheads). The z-stack spanned the thickness of the ganglion and both dorsal and ventral cells are shown in the projection. Calibration bar = 50 μm, all panels.</p

Carboxylic Acids as Biomarkers of Biomphalaria alexandrina Snails Infected with Schistosoma mansoni

Biomphalaria alexandrina snails play an indispensable role in transmission of schistosomiasis. Infection rates in field populations of snails are routinely determined by cercarial shedding neglecting prepatent snail infections, because of lack of a suitable method for diagnosis. The present study aimed at separation and quantification of oxalic, malic, acetic, pyruvic, and fumaric acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to test the potentiality of these acids to be used as diagnostic and therapeutic biomarkers. The assay was done in both hemolymph and digestive gland-gonad complex (DGG) samples in a total of 300 B. alexandrina snails. All of the studied acids in both the hemolymph and tissue samples except for the fumaric acid in hemolymph appeared to be good diagnostic biomarkers as they provide not only a good discrimination between the infected snails from the control but also between the studied stages of infection from each other. The most sensitive discriminating acid was malic acid in hemolymph samples as it showed the highest F-ratio. Using the Z-score, malic acid was found to be a good potential therapeutic biomarker in the prepatency stage, oxalic acid and acetic acid in the stage of patency, and malic acid and acetic acid at 2 weeks after patency. Quantification of carboxylic acids, using HPLC strategy, was fast, easy, and accurate in prediction of infected and uninfected snails and possibly to detect the stage of infection. It seems also useful for detection of the most suitable acids to be used as drug targets