Research on the Mechanism of Metaldehyde on Pomacea canaliculata.

Golden apple snail (Pomacea canaliculata), a major alien invasive organism in China, affects food production and poses a threat to human health. Metaldehyde is a highly effective, commonly used snail killer with low toxicity. Virulence determination, tissue section, iTRAQ and RNA interference were used to systematically study the toxicity of metaldehyde on P. canaliculata. The molluscicidal activity tests showed that metaldehyde exhibits strong toxicity against P. canaliculata. Physiological and biochemical data indicate that metaldehyde can cause damage to the gills, liver, pancreas, and kidneys of snails, also reduce the oxygen consumption rate and ammonia excretion rate of golden apple snails, and cause neurological diseases. The proteome of the gill region of the golden apple snail after exposure to metaldehyde was analyzed by using iTRAQ technology. A total of 360 differential proteins were identified, and four target proteins were screened, namely, alpha-protein kinase 1 (ALPK1), cubilin (CUBN), sodium- and chloride-dependent GABA transporter 2 (GAT2), and acetylcholinesterase (AChE). RNAi was used to target the four proteins. After the ALPK1 and CUBN protein genes were interfered with by metaldehyde treatment, it was found that the mortality rate of the golden apple snail significantly increased. However, interference of GAT2 and AChE protein genes by metaldehyde led to no significant change in the mortality rates of the snails. The histopathological observation of the gill showed that the rate of cilia shedding in the gill decreased after the interference of ALPK1 and CUBN protein genes.

Molluscicidal activity of polyhexamethylene biguanide hydrochloride on the early-life stages and adults of the Biomphalaria glabrata (Say, 1818).

Schistosomiasis is a disease that affects tropical and subtropical areas and is considered the second most prevalent parasitic disease in the world. One of the ways of combating this disease is the use of molluscicidal agents to eliminate or reduce the population of intermediate host snails. Polyhexamethylene biguanide hydrochloride (PHMB) is a chemical biocide commonly used as a disinfectant and antiseptic in the food industry and very successfully for the disinfection of swimming pools. The US Environmental Protection Agency (USEPA) indicated the PHMB as low environmental risk. The present study aimed to evaluate the molluscicidal activity of the PHMB in freshwater snail (Biomphalaria glabrata), intermediate host of Schistosoma mansoni. The PHMB showed high toxicity against all stages of the snail B. glabrata: embryos, new-borns and adults. The LC50 estimated was 0.98 mg L-1; 1.43 mg L-1 and 1.49 mg L-1, respectively, after exposure of 144 h for embryos and 96 h for new-borns and adults. PHMB did not prevent the development of embryos within the egg mass, since at all concentrations evaluated 80% of the embryos managed to develop until the hypo-stage, which is the last stage of development before hatching. However, PHMB inhibited the hatching of embryos by 100% at all concentrations above 1.6 mg L-1. PHMB proved to be a promising substance in the fight against schistosomiasis by eliminating the intermediate host (B. glabrata). This was the first study that makes an experimental observation of the molluscicidal activity of PHMB.

Isolation and characterization of metaldehyde-degrading bacteria from domestic soils.

Metaldehyde is a common molluscicide, used to control slugs in agriculture and horticulture. It is resistant to breakdown by current water treatment processes, and its accumulation in drinking water sources leads to regular regulatory failures in drinking water quality. To address this problem, we isolated metaldehyde-degrading microbes from domestic soils. Two distinct bacterial isolates were cultured, that were able to grow prototrophically using metaldehyde as sole carbon and energy source. One isolate belonged to the genus Acinetobacter (strain designation E1) and the other isolate belonged to the genus Variovorax (strain designation E3). Acinetobacter E1 was able to degrade metaldehyde to a residual concentration < 1 nM, whereas closely related Acinetobacter strains were completely unable to degrade metaldehyde. Variovorax E3 grew and degraded metaldehyde more slowly than Acinetobacter E1, and residual metaldehyde remained at the end of growth of the Variovorax E3 strain. Biological degradation of metaldehyde using these bacterial strains or approaches that allow in situ amplification of metaldehyde-degrading bacteria may represent a way forward for dealing with metaldehyde contamination in soils and water.

Chemical compounds toxic to invertebrates isolated from marine cyanobacteria of potential relevance to the agricultural industry.

In spite of advances in invertebrate pest management, the agricultural industry is suffering from impeded pest control exacerbated by global climate changes that have altered rain patterns to favour opportunistic breeding. Thus, novel naturally derived chemical compounds toxic to both terrestrial and aquatic invertebrates are of interest, as potential pesticides. In this regard, marine cyanobacterium-derived metabolites that are toxic to both terrestrial and aquatic invertebrates continue to be a promising, but neglected, source of potential pesticides. A PubMed query combined with hand-curation of the information from retrieved articles allowed for the identification of 36 cyanobacteria-derived chemical compounds experimentally confirmed as being toxic to invertebrates. These compounds are discussed in this review.

Systematic review of pentachlorophenol occurrence in the environment and in humans in China: not a negligible health risk due to the re-emergence of schistosomiasis.

Pentachlorophenol (PCP) has been widely used for killing snails in areas of China where schistosomiasis is epidemic. With the re-emergence of schistosomiasis, the warranted production and consumption of PCP has inevitably resulted in persistent environmental contamination by it and its impurities, polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). This study aimed to evaluate the contamination status and human burden of PCP and its impurities (PCDD/Fs) in China, considering the previous epidemic and re-emergence of schistosomiasis. We searched studies relevant to PCP occurrence in the environment and in humans in China. Data on snail elimination areas were included to estimate PCP consumption. Relevant publications were analyzed to distinguish PCDD/Fs contamination from PCP usage. PCP contamination was detected ubiquitously in various environmental media and in human samples; environmental levels were generally low, with the exception of some hot spots. In schistosomiasis-epidemic areas, there were significantly higher PCP levels in the environment and in humans than in control areas. Spatial disparities indicated the consistency between serious schistosomiasis epidemic areas and hot spots of PCP contamination. The data suggest an increased trend in PCP contamination of the environment. Specific PCDD/Fs contamination from PCP usage existed even at low environmental levels. The occurrence of PCP in the environment and in humans positively correlated with the epidemic of schistosomiasis. Thyroid-disrupting effects and cancer risk caused by PCP and PCDD/Fs even at low environmental levels in China's schistosomiasis-epidemic areas are of concern.

Determination and estimation of partitioning properties for substituted phosphates and thiophosphates.

Substituted phenylglyoxylonitrile oximino phosphates and thiophosphates invested as a synergist of molluscicide killing snail eggs have been exposed to the environment with very little attention. The partitioning properties, aqueous solubility (S(w)), n-octanol-water partition coefficient (K(ow)) and soil organic carbon sorption coefficients (K(oc)) of 16 O, O'-dialkyl, O''-(substituted phenylglyoxylonitrile oximino) phosphates and thiophosphates, were determined by the traditional shaking flask method. The parameters of molecular fragment connectivity indices (MFCIs) and linear solvation energy relationships (LSERs) were used as molecular descriptors to establish a series of correlation equations successfully. The obtained correlation equations provided a quantitative method to predict the three partitioning properties for new exploited substituted phosphates and thiophosphates. More valuable, the successful application of MFCIs provides us with a good example and a good idea to improve traditional molecular connectivity indices (MCIs).

Alkyl, aryl, alkylarylquinoline, and related alkaloids.

The Rutaceae continues to be the primary source of new alkyl-, aryl-, and alkylarylquinolin/ones. In the past 17 years, the overall distribution of these alkaloid types within the family has changed little since the chemosystematics reviews by Waterman (270), Mester (40), and da Silva et al. (279). Alkylquinolones dominate the reported isolations with about 51% of the total, with arylquinolones (16%), alkylquinolines (15%), alkylarylquinolines (11%), arylquinolines (3%), alkylarylquinolones (2%), and quinolines (2%) as the significant structural groups contributing to the remainder of this class of alkaloids. The alkyl-, aryl-, and alkylarylquinolin/one alkaloids occur in 50 species belonging to 24 genera and 6 subfamilies. Despite the intensive chemical exploration of many species from other plants in the Rutales family, but not in the family Rutaceae, the first alkaloid alkylquinolone from a simaroubaceous plant (160) was not reported until 1997. Although many additional alkaloids have been reported, some of new structural types (Bo.4), substantial biosynthetic work on plant-derived alkylquinolin/ones has not yet been carried out. The biosynthesis of some of these alkaloids in bacteria was firmly established as being derived from anthranilic acid. Outside of the Rutales, alkyl-, aryl-, and alkylarylquinolin/ones have not been found, except for simple quinoline (A.1; only one) and 2-methylquinoline derivatives in the Zygophyllaceae, and only an atypical quinolone derivative (Ao.1) in the Asteraceae family. A few 3-phenylquinolines (2), 3-(1H-indol-3-yl)quinoline (1), and quinoline-quinazoline (1) alkaloids have been reported from only a single genus in the Zygophyllaceae. Tryptophan-derived quinolines in higher plants are confined to a few 2-carboxylicquinolin/ones (6) and 4-carbaldehydequinolines (5); the former found in the Ephedraceae (5), Boraginaceae (1), Fagaceae (1), Ginkgoaceae (1), Plumbaginaceae (1), Solanaceae (1), and Apiaceae (1), and the latter in the Moraceae (3), Alliaceae (1), and Pontederiacae (1). The number of quinolones derived from glycine and a polyketide is also limited. 5-Alkyl-2-methylquinolin-4(1H)-ones (8) occur in the Euphorbiaceae, and 5-alkyaryl-2-methylquinolin-4(1H)-ones ((3) in the Sterculiaceae. Alkylquinolin/ones are well-known as typical alkaloids of three Proteobacteria and three Actinobacteria; the genus Pseudomonas yielded the majority (46%) of the total number of alkaloids reported (39). 2-Carboxylicquinolin/ones (4) and 4-carbaldehydequinolines (6) are minor constituents in both divisions of bacteria. More interesting are the quinolactacins (7), in which the second nitrogen is derived from L-valine or L-isoleucine, recently reported to occur only in the fungus Penicillium. Many of these diverse alkaloids have served directly as medicines or as lead compounds for the synthesis (258) of derivatives with an improved biological profile. It is apparent from the summary view of the alkyl-, aryl-, and alkylarylquinolin/ones reported in the Rutaceae that they help to confirm the affinity between Rutoideae tribes and provide firm support for placing the Spathelioideae and the Dictyolomatoideae close to the more primitive Zanthoxyleae tribe. On the other hand, the bacteria and fungi are needed for more substantial chemical studies. When more data become available, it is likely that useful systematic correlations will emerge. More detailed studies regarding the biosynthetic pathways of the alkyl-, aryl-, and alkylarylquinolin/ones in the Rutaceae and in bacteria are needed. Such studies would clarify the differences in the pathways based on their derivation from anthranilic acid in bacteria and in rutaceous plants. Finally, this survey indicates that the Rutaceae, and various bacterial and fungal species offer considerable potential for the discovery of new or known alkaloids with significant and possibly valuable biological activities.

Molluscicidal activity of vulgarone B against ram's horn snail (Planorbella trivolvis).

The ram's horn snail (Planorbella trivolvis (Say)) is an intermediate host for a digenetic trematode (Bolbophorus confusus (Krause) Dubois) that has recently been discovered to be a significant problem in commercial channel catfish (Ictalurus punctatus Raf) production ponds in the Mississippi Delta region in the USA. In these catfish ponds, the digenetic life cycle of this parasitic trematode involves two intermediate hosts, the ram's horn snail and the channel catfish, and the final host, the American white pelican (Pelecanus erythrorhynchos Gmelin). One approach to eradicate this problem is to disrupt the life cycle of the parasitic trematodes by eliminating the snails. During our search for natural-product-based molluscicides to control the snails in the catfish ponds, vulgarone B, isolated from the steam distillate of the aerial parts of the plant Artemisia douglasiana Besser (Asteraceae), was found to be active towards the snails with a LC50 of ca 24 microM. Channel catfish toxicity studies indicated a LC50 of ca 207 microM. Vulgarone B may be an environmentally acceptable alternative for snail control in aquaculture.

Community participation as an interactive learning process: experiences from a schistosomiasis control project in Zimbabwe.

A schistosomiasis control project was undertaken from 1994 to 1998 in Guruve District, Zimbabwe, based on the active involvement of local communities in the growing and application of the molluscicidal plant Phytolacca dodecandra as a supplement to other control measures such as chemotherapy and health education. The berries of P. dodecandra are highly molluscicidal to the intermediate host snails of schistosomiasis and is rapidly degradable in water. It was observed that plant care fluctuated during the study period. Only a few households participated in snail control activities, although 97% of respondents in a baseline survey had stated a willingness to take part. This invoked an investigation to explore the reality from a user perspective. Focus group discussions, semi-structured interviews and observations were used to solicit information. In spite of a cultural inclination for collective work, many reasons for low-level involvement emerged, inter alia the low perceived value of the project, demands for tangible benefits, inaccessible fields and weak leadership. This study shows that community participation is a complex process upon which a multiplicity of social and cultural determinants have an impact. If community participation is to become successful in development programmes it ought to be viewed as a mutual learning process where obstacles are identified and discussed and solutions shared among community members and project staff.

Molluscicidal and antifungal activity of Erigeron speciosus steam distillate.

The steam-distilled fraction of the aerial parts of Erigeron speciosus (Lindl) DC was tested for activity against strawberry plant pathogenic fungi Botrytis cinerea Pers ex Fr, Colletotrichum acutatum Simmonds, C fragariae Brooks, C gloeosporioides (Penz) Penz & Sacc, and the intermediate host snail Planobdella trivolvis that harbors the trematode, Bolbophorus confusus, that infests and causes severe infections in pond-raised catfish in the Mississippi Delta region of the USA. Bioautography on silica TLC plates demonstrated antifungal activity in the steam distillate. Preliminary bioassays of the steam distillate indicated the presence of phytochemicals toxic to P trivolvis. The bioactive compounds methyl 2Z, 8Z-deca-2,8-diene-4,6-diynoate and its 2E, 8E isomer were isolated by bioassay-guided fractionation and chromatographic techniques and identified by 1H NMR spectroscopy.

Bioactivity of Anagallis arvensis and Calendula micrantha plants, treated with ammonium nitrate, superphosphate and potassium sulphate fertilizers, on Biomphalaria alexandrina.

The survival rate of B. alexandrina snails maintained in aqueous solutions of the two tested plants (Calendula micrantha and Anagallis arvensis) decreased gradually with time until the 9th week and 10th week where the survival rate was zero in the high concentration of A. arvensis and C. micrantha, respectively, meanwhile, the survival rate of the control was 20%. Also, the two plants caused reduction in hatchability of snails egg masses. Thus, the percent of hatching in A. arvensis (82 ppm) was 46% and in C. micrantha was 72% compared with control (97.29%). Both plants reduced the infection rate of Biomphalaria alexandrina snails with Schistosoma mansoni miracidia to 41.17% and 61.9%, respectively, compared with control (90%). C. micrantha caused much higher reduction in snail infection rate than A. arvensis. The prepatent period was significantly prolonged in snails maintained at higher concentration of both plants. The cercarial output (expressed as mean number/snail) revealed that, A. arevensis caused a significant reduction in cercarial production than control. While, high concentration (120 ppm) of C. micrantha caused a significant elevation in the mean number of cercariae/snail. However, the total number of cercariae produced by all snails in each group showed a reverse relation with the tested concentrations in both plants.

Biodegradability of the molluscicidal saponins of Phytolacca dodecandra.

The biodegradability of water-extracted saponins of berries from the Endod plant, Phytolacca dodecandra L'Herit, was evaluated under OECD standardized conditions. Persistence of the saponins was evaluated by determination of saponin concentrations in water over a 30-day period, using a quantitative HPLC method and a semiquantitative hemolytic assay, which is considered a potential field method. The two methods were compared. Bioassays were simultaneously conducted using Biomphalaria glabrata fresh water snails to assess the molluscicidal potency over time. All experiments were carried out in water, which was chemically balanced to sustain snails, and to one set river water was added to the medium to mimic natural conditions. (1) Saponin concentrations in water treated with an aqueous extract of P. dodecandra cultivar E44 were stable for 2 days then rapidly decreased during the third and fourth day. Lethal concentrations to snails were LC50 = 9.6 mg/l (95% CI: 6.3 to 19.4) in one set and LC50 = 6.8 mg/l (95% CI: 5.4 to 12.2) in the other. Adding river water to one set of the experiments had no effect on the mean saponin concentration over time (F = 0.02, P = >0.05). (2) Comparison of the hemolytic assay with the HPLC method showed no significant difference in mean saponin concentrations (t = 0.32, P > 0.05, 95% CI: -2.67 to 3.64), and a correlation of r2 = 0.88 between the two methods. (3) The saponin fraction of an aqueous extract of P. dodecandra was readily biodegraded (t1/2 = 15.8 h), and the complete consumption within a 10-day window indicates ready degradability in aquatic environments under aerobic conditions. (4) These results show that the use of Endod berries for snail control in schistosomiasis-infested water bodies is environmentally acceptable.

New possible molluscicides from Calendula micrantha officinalis and Ammi majus. II. Molluscicidal, physiological, and egg-laying effects against Biomphalaria alexandrina and Bulinus truncatus.

In the present study, the effects of CuSo4 and crude extracts of the different parts of Calendula micrantha officinalis and Ammi majus, i. e., leaves, stems, roots, and flowers, on adult Biomphalaria alexandrina and Bulinus truncatus were investigated. Generally, leaves and flowers of both plants exhibited marked potency in killing the snail vectors of schistosomiasis. The recorded LC50 and LC90 values showed that C. officinalis was more toxic to both snails than A. majus, and B. truncatus are more sensitive to the extracts of both plants than B. alexandrina. Snails that are produced from snails previously exposed to low doses were more sensitive to the tested extracts, which may give primary indication of no possibility of inherited resistance. Moreover, prolonged exposure to the sublethal concentrations of A. majus have a definite lethal effect on the egg laying and longevity of both snails. Also, treatment with sublethal doses of both plants clearly inhibited the transaminase activity (ALAT, ASAT), diminished the total protein content, and increased markedly total lipid contents in the hemolymph of both snails.

Tumor promoter-like activity of the molluscicidal latex of 'Crown-of-Thorns' (Euphorbia milii var. hislopii) in the V79 metabolic cooperation assay.

The latex of 'Crown-of-Thorns' (Euphorbia milii var. hislopii, syn. E. splendens) has been shown to be a potent plant molluscicide that could be used against the snails which are intermediate hosts of Schistosoma trematodes. However, a comprehensive toxicological evaluation of the latex is necessary before its large-scale use in schistosomiasis control becomes possible. In fact, one cause for concern is the presence of tumor-promoting phorbol esters in several plants of the Euphorbiaceae family. Phorbol esters as well as a number of other known tumor promoters share the common property of inhibiting metabolic cooperation (i.e., exchange of low molecular weight molecules via gap junctions) between Chinese hamster V79 cells in monolayer cultures. The present study was undertaken to determine if latex of E. milii presents tumor promoter-like activity is this short-term in vitro assay. Samples of lyophilized E. milii latex were tested at a noncytotoxic concentration range (1, 10, 50 and 100 micrograms/ml) in three independent experiments. 12-O-Tetradecanoylphorbol-13-acetate (10 ng/ml) was used as positive control. In all three assays, E. milii latex consistently inhibited metabolic cooperation between V79 cells at concentrations > or = 10 micrograms/ml. These results that E. milii latex contains tumor-promoting substances. These findings suggest that the use of crude latex as a molluscicide may pose a carcinogenic hazard to people who are continuously exposed to the product.

Tumor promoter-like activity of the molluscicidal latex of 'Crown-of-Thorns' (Euphorbia milii var. hislopii) in the V79 metabolic cooperation assay

The latex of 'Crown-of-Thoms'(Euphorbia miliivar. hislopii, syn. E. splendens) has been shown to be a potent plant molluscicide that could be used against the snails which are intermediate hosts of Schistosoma trematodes. However, a comprehensive toxicological evaluation of the latex is necessary before its large-scale use in schistosomiasis control becomes possible. In fact, one cause for concern is the presence of tumor-promoting phorbol esters in several plants of the Euphorbiaceae family. Phorbol esters as well as a number of other known tumor promoters share the common property of inhibiting metabolic cooperation (i.e., exchange of low molecular weight molecules via gap junctions) between Chinese hamster V79 cells in monolayer cultures. The present study was undertaken to determine if latex of E. milii presents tumor promoter-like activity in this shortterm in vitro assay. Samples of lyophilized E. milii latex were tested at a noncytotoxic concentration range (1, 10, 50 and 100 mug/ml) in three independent experiments. 12-0-Tetradecanoylphorbol-13-acetate (10 ng/ml) was used as positive control. In all three assays, E. milii latex consistently inhibited metabolic cooperation between V79 cells at concentrations (10 mug/ml. These results indicate that E. milii latex contains tumor-promoting substances. These findings suggest that the use of crude latex as a molluscicide may pose a carcinogenic hazard to people who are continuously exposed to the product.

Fermentation and toxin studies of the molluscicidal strains of Bacillus brevis.

Strain SS86-4 was one of 40 Bacillus brevis strains shown to be molluscicidal to the schistosomiasis snail vector Biomphalaria glabrata. When grown in mB4 medium in 2-L fermentors, SS86-4 was molluscicidal only if fructose or phenylalanine was present in the medium. This is reminiscent of secondary fermentation factor effects, in this case an antioxidant effect. In vivo proteases also were capable of reducing molluscicidal activity. The molluscicidal toxin has an LC50 of 1 microgram toxin protein ml-1 (approx. 1 p.p.m.) and may be described as a small proteinaceous, heat-stable, oxygen-sensitive entity associated with the particulate portion of the cell wall fraction of B. brevis that is formed prior to sporulation. Initial information indicates that its HPLC signature shows major peaks at 148.37 and 163.96 s and consists of two bands of approximately 5.3 kDa and 8.7 kDa on PAGE gel.

Investigation of the diurnal, ontogenetic and seasonal variation in the molluscicidal saponin content of Phytolacca dodecandra aqueous berry extracts.

Daily variation in the content of the saponins of the berries of Phytolacca dodecandra was determined in berries harvested at the full grown green unripe stage of development once every two hours. The saponin content was quantitatively determined by HPLC analysis of the molluscicidal monodesmosidic saponins in the aqueous extract of the crushed berries. No significant daily variations were observed in extracts of berries collected at different times of day (P > 0.05 by ANOVA). But, seasonal variation was apparent in extracts prepared from berries harvested from the same plants once every two weeks. Berries of highest saponin content were those harvested during the dry season and just before the onset of the rainy season. The seasonal variation was attributed to increased water stress during the dry season. Very young racemes had the highest saponin content, while mature berries produced the lowest saponin concentration. There was, however, consistency in the berry saponin composition during the day, season and all stages of berry development.

Studies on antibiotic 230 as a molluscicide.

In the course of studying molluscicidal effects of microorganisms, a microorganism belonging to Streptomycetaceae, Streptomyces griseolus (Strain 230) was isolated from soil and a certain microbial ingredient proved to have a molluscicidal activity. The effective substance was named antibiotic 230. As a result of several investigations on the antibiotic, the following chemical features were made clear. The chemical substance was an indol-carbazole-heterocyclic compound, the molecular weight 446 and the molecular formula C28H26N4O3. The antibiotic demonstrated a molluscicidal effect against Oncomelania snails and their eggs both in the laboratory and in the field. The compound had a wide bacteriostasis spectrum and a slight toxicity to fish in addition to the above, but it was non-toxic to plants.