Prey-mediated effects of Mpp51Aa2-producing cotton on longevity and reproduction of Orius majusculus.

Genetically engineered (GE) cotton event MON 88702, producing Mpp51Aa2 (previously mCry51Aa2) from Bacillus thuringiensis (Bt), controls sucking pests, such as Lygus spp. (Hemiptera: Miridae) and thrips (Thysanoptera). Ingesting high doses of the insecticidal protein resulted in adverse effects on life table parameters of beneficial, predatory Orius spp. (Hemiptera: Anthocoridae). This triggered laboratory studies with more realistic food treatments, including different combinations of prey types with and without Bt protein to further characterize risks to this important group of non-target organisms. In this work, exclusive feeding of frozen spider mites (Tetranychus urticae, Acari: Tetranychidae) from Bt cotton confirmed adverse effects on longevity and fecundity of O. majusculus adults. Alternate feeding of Bt protein-containing spider mites and Bt-free Ephestia kuehniella (Lepidoptera: Pyralidae) eggs mitigated effects on longevity, but not on fecundity. When living larvae of Spodoptera littoralis (Lepidoptera: Noctuidae) from Bt cotton were fed to the predators, however, no effects on longevity and reproduction of female O. majusculus were observed, despite the fact that Bt protein concentrations in larvae were almost as high as concentrations in spider mites. When a diverse mix of prey species with various Bt protein concentrations is consumed in the field, it is unlikely that exposure of Orius spp. to Mpp51Aa2 is high enough to exert adverse effects on predator populations. MON 88702 cotton may thus be a valuable tool for integrated management of sucking pests.

Further pathogenicity testing of Verticillium nonalfalfae, a biocontrol agent against the invasive Tree of Heaven (Ailanthus altissima), on non-target tree species in Europe.

Verticillium nonalfalfae is an effective biological control agent against the highly invasive Tree of Heaven (Ailanthus altissima) in Europe, and previous research on ten economically and ecologically important tree species occurring in Austria have so far not revealed undesired non-target effects. In this study, another nine tree species including five native, two non-native as well as two invasive alien tree species were tested for susceptibility to the particular strain of V. nonalfalfae (Vert56) used for biological control of A. altissima. Stem inoculations on potted seedlings revealed that this strain of V. nonalfalfae is generally host-adapted to A. altissima. It induced chlorosis, necrosis and wilting already within two weeks post inoculation on A. altissima and resulted in almost completely defoliated or dead seedlings at the end of the vegetation period. Apart from two species (Quercus rubra and Sorbus aucuparia), that suffered from other abiotic/biotic agents, no mortality was observed on all other tree species tested; however, symptoms caused by other abiotic factors were also found on Prunus avium and Ulmus glabra. All tested tree species exhibited vascular discolorations and the fungus could be re-isolated at varying frequencies (6-100%) from inoculated seedlings of all non-target tree species, although five of these species exhibited no external symptoms. Results confirmed high susceptibility (S) of A. altissima to V. nonalfalfae, whereas Acer platanoides, Castanea sativa, Q. rubra, S. aucuparia and U. glabra were considered as tolerant (T), and A. negundo, P. avium, P. serotina and Q. petraea were rated as possible resistant (PR) due to the low rates of re-isolation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12600-022-01032-z.

Assessing Effects of Genetically Modified Plant Material on the Aquatic Environment Using higher-tier Studies.

Genetically modified organisms are used extensively in agriculture. To assess potential side effects of genetically modified (GM) plant material on aquatic ecosystems, only a very small number of higher-tier studies have been performed. At the same time, these studies are particularly important for comprehensive risk assessment covering complex ecological relationships. Here we evaluate the methods of experimental higher-tier effect studies with GM plant material (or Bt toxin) in comparison to those well-established for pesticides. A major difference is that nominal test concentrations and thus dose-response relationships cannot easily be produced with GM plant material. Another important difference, particularly to non-systemic pesticides, is that aquatic organisms are exposed to GM plant material primarily through their feed. These and further differences in test requirements, compared with pesticides, call for a standardisation for GM-specific higher-tier study designs to assess their potentially complex effects in the aquatic ecosystems comprehensively.

Non-target effects of agri-environmental schemes on solitary bees and fungi in the United Kingdom.

Agri-environmental schemes (AES) are used to enhance pollinator diversity on agricultural farms within the UK. Though the impacts of these schemes on archetypal pollinator species such as the bumblebee (Bombus) and honeybee (Apis) are well-studied, the effects on non-target bee species like solitary bees, in the same environment, are generally lacking. One goal of AES is to alter floral provision and taxonomic composition of plant communities to provide better forage for pollinators, however, this may potentially impact other ecological communities such as fungal diversity associated with plant-bee communities. Fungi are integral in these bee communities as they can impact bee species both beneficially and detrimentally. We test the hypothesis that alteration of the environment through provision of novel plant communities has non-target effects on the fungi associated with solitary bee communities. We analyse fungal diversity and ecological networks formed between fungi and solitary bees present on 15 agricultural farms in the UK using samples from brood cells. The farms were allocated to two categories, low and high management, which differ in the number of agri-environmental measures implemented. Using internal transcribed spacer metabarcoding, we identified 456 fungal taxa that interact with solitary bees. Of these, 202 (approximately 44%) could be assigned to functional groups, the majority being pathotrophic and saprotrophic species. A large proportion was Ascosphaeraceae, a family of bee-specialist fungi. We considered the connectance, nestedness, modularity, nestedness overlap and decreasing fill, linkage density and fungal generality of the farms' bee-fungi ecological networks. We found no difference in the structure of bee-fungi ecological networks between low and high management farms, suggesting floral provision by AES has no significant impact on interactions between these two taxonomic groups. However, bee emergence was lower on the low management farms compared to high management, suggesting some limited non-target effects of AES. This study characterizes the fungal community associated with solitary bees and provides evidence that floral provision through AES does not impact fungal interactions.

Addressing the challenges of non-target feeding studies with genetically engineered plant material - stacked Bt maize and Daphnia magna.

Previous studies reported adverse effects of genetically engineered maize that produces insecticidal Cry proteins from Bacillus thuringiensis (Bt) on the water flea Daphnia magna. In the current study, effects of flour, leaves, or pollen from stacked Bt maize that contains six Bt proteins (SmartStax) in two plant backgrounds on life table parameters of D. magna were investigated. Adverse effects were observed for Bt maize flour, originating from different production fields and years, but not for leaves or pollen, produced from plants grown concurrently in a glasshouse. Because leaves contained eight to ten times more Cry protein than flour, the effects of the flour were probably not caused by the Cry proteins, but by compositional differences between the plant backgrounds. Furthermore, considering the natural range of variation in the response of D. magna to conventional maize lines, the observed effects of Bt maize flour were unlikely to be of biological relevance. Our study demonstrates how Cry protein effects can be separated from plant background effects in non-target studies using Bt plant material as the test substance and how detected effects can be judged for their biological relevance.

Toxicological risk assessment of some commonly used insecticides on Cotesia flavipes, a larval parasitoid of the spotted stem borer Chilo partellus.

Cotesia flavipes Cameron is an important larval parasitoid exploited for the control of the spotted stem borer, Chilo partellus (Swinhoe). Several studies have evaluated the toxic effects of insecticides on C. partellus, however, little is known about non-target effects of insecticides on this parasitoid, when used to control C. partellus. This laboratory study evaluated the lethal and sublethal effects of twelve insecticides on C. flavipes. Residual toxicity tests showed that organophosphates (chlorpyrifos, triazophos and profenofos) exhibited highest contact toxicity to C. flavipes adults with a LC50 range from 0.63 to 1.05 mg a.i/l, while neonicotinoids (nitenpyram, acetamiprid and imidacloprid) were less toxic to C. flavipes with a LC50 range from 1.27 to 139.48 mg a.i/l. Sugar-insecticide feeding bioassays showed that organophosphates, pyrethroids (cypermethrin, bifenthrin and lambda-cyhalothrin) and carbamates (thiodicarb, carbaryl and methomyl) were highly toxic to C. flavipes adults and caused 100% mortality at 48 h of exposure, while imidacloprid caused 66% mortality at 48 h of exposure. Risk quotient analysis showed that among all tested insecticides, imidacloprid and acetamiprid were less toxic to C. flavipes adults with a risk quotient value of 0.88 and 1.6, respectively. Furthermore, exposure of immature C. flavipes through their host bodies significantly decreased the parasitism rate at their F1 and F2 generations. Risk quotient analysis of insecticides indicated that imidacloprid and acetamiprid were the least toxic to C. flavipes. This study provides important information that will be used in incorporating the most suitable insecticides in integrated pest management programs with reduced negative impacts on non-target beneficial arthropods.

Prevalence and parasite load of nematodes and trematodes in an invasive slug and its susceptibility to a slug parasitic nematode compared to native gastropods.

The invasive slug Arion vulgaris (Gastropoda: Arionidae) is an agricultural pest and serious nuisance in gardens of Central and Northern Europe. To investigate if the success of A.vulgaris in Norway can be attributed to a release from parasites, we compared the prevalence and parasite load of nematodes and trematodes in A. vulgaris to that of three native gastropod species, A. circumscriptus, A. fasciatus and Arianta arbustorum, in SE Norway. We found A. vulgaris to have the highest prevalence of both parasite groups (49% nematodes, 76% trematodes), which does not support the parasite release hypothesis, but rather points to A. vulgaris as a potentially important intermediate host of these parasites. For trematodes the number of individuals (parasite load) did not differ among host species; for nematodes it was higher in A. vulgaris than A. fasciatus. To further compare the parasite susceptibility of the surveyed gastropods, we exposed A. vulgaris, A. fasciatus, and A. arbustorum to a slug parasitic nematode, Phasmarhabditis hermaphrodita, in the laboratory. This nematode is commercially available and widely used to control A. vulgaris. The non-target species A. fasciatus was most affected, with 100% infection, 60% mortality and significant feeding inhibition. A. vulgaris was also 100% infected, but suffered only 20% mortality and little feeding inhibition. The load of P. hermaphrodita in infected specimens was not significantly different for the two Arion species (median: 22.5 and 45, respectively). Only 35% of A. arbustorum snails were infected, none died, and parasite load was very low (median: 2). However, they showed a near complete feeding inhibition at highest nematode dose, and avoided nematode-infested soil. Our results indicate that A. vulgaris may be less susceptible to P. hermaphrodita than the native A. fasciatus, and that non-target effects of applying this nematode in fields and gardens should be further investigated.

Bacillus thuringiensis protein Vip3Aa does not harm the predator Propylea japonica: A toxicological, histopathological, biochemical and molecular analysis.

The ladybeetle Propylea japonica is a widely distributed natural enemy in many agricultural systems. P. japonica is often used as a test organism for safety assessments of transgenic Bacillus thuringiensis crops. Plant varieties expressing the Vip3Aa insecticidal protein are not currently commercially available in China. In this study, protease inhibitor E-64 was used as a positive control to examine the responses of P. japonica larvae to a high concentration of Vip3Aa proteins. Larvae that were fed E-64 had increased mortality and prolonged developmental period, but these parameters were unaffected when larvae were fed Vip3Aa. The epithelial cells of midguts were intact and closely connected with the basal membrane when larvae were fed Vip3Aa, but the epithelial cells degenerated in the E-64 treatment. The activities of antioxidative enzymes and expression levels of detoxification-related genes in P. japonica larvae were not altered after exposure to Vip3Aa; however, these biochemical and molecular parameters were significantly changed in the E-64 treatment. The results demonstrate that Vip3Aa protein is not harmful to the predator P. japonica.

Changes in Larval Mosquito Microbiota Reveal Non-target Effects of Insecticide Treatments in Hurricane-Created Habitats.

Ephemeral aquatic habitats and their associated microbial communities (microbiomes) play important roles in the growth and development of numerous aquatic insects, including mosquitoes (Diptera). Biological control agents, such as Bacillus thuringiensis israelensis (Bti) or insect growth regulators (e.g., methoprene), are commonly used to control mosquitoes in these habitats. However, it is unknown how commonly used control compounds affect the mosquito internal microbiome and potentially alter their life history traits. The objectives of this study were threefold: characterize the internal microbiota of Aedes larvae (Culicidae) in ephemeral forested mosquito habitat using high-throughput amplicon based sequencing, assess how mosquito control treatments affect the internal microbial communities of larval mosquitoes, and determine if changes to the microbiome resulted from direct or indirect treatment effects. The larval microbiome varied in community composition and diversity with development stage and treatment, suggesting potential effects of control compounds on insect microbial ecology. While microbial community differences due to Bti treatment were a result of indirect effects on larval development, methoprene had significant impacts on bacterial and algal taxa that could not be explained by indirect treatment effects. These results provide new information on the interactions between pesticide treatments and insect microbial communities.

Effects of straw leachates from Cry1C-expressing transgenic rice on the development and reproduction of Daphnia magna.

The transgenic rice line T1C-19 provides high resistance to lepidopteran pests because of the synthesis of the Bacillus thuringiensis (Bt) insecticidal protein Cry1C. It thus shows good prospect for commercial planting in China. Species of Cladocera, an order of aquatic arthropods commonly found in aquatic ecosystems such as rice paddies, might be exposed to the insecticidal protein released from Bt-transgenic rice-straw residues. For the study reported herein, we used Daphnia magna (water flea) as a representative of Cladocera to evaluate whether aquatic arthropods are adversely affected when exposed to Bt rice-straw leachates. We exposed D. magna to M4 medium containing various volume percentages of medium that had been incubated with T1C-19 rice straw or rice straw from its non-transformed near-isoline Minghui 63 (MH63) for 21 days. Compared with pure M4 medium (control), the fitness and developmental and reproduction parameters of D. magna decreased significantly when exposed to rice-straw leachates; conversely, no significant differences between the T1C-19 and MH63 rice-straw leachate treatments were observed, indicating that the Bt rice straw leachate did not adversely affect this non-target species.

Safety of Bacillus thuringiensis Cry1C protein for Daphnia magna based on different functional traits.

Cry1C is a Bacillus thuringiensis (Bt) insecticidal protein and it can be produced by transgenic rice lines developed in China. Cladocera species are common aquatic arthropods that may be exposed to insecticidal proteins produced in Bt-transgenic plants through ingestion of pollen or crop residues in water. As the cladoceran Daphnia magna plays an important role in the aquatic food chain, it is important to assess the possible effects of Bt crops to this species. To evaluate the safety of the Cry1C protein for D. magna, individuals were exposed to different concentrations of purified Cry1C protein in M4 medium for 21 days. Potassium dichromate (K2Cr2O7), a known toxicant to D. magna, was added to M4 medium as a positive control treatment, and pure M4 medium was used as a negative control. Our results show that developmental, reproductive, and biochemical parameters of D. magna were not significantly different between Cry1C and negative control treatments but were significantly inhibited by the positive control. We thus conclude that D. magna is insensitive to Cry1C.

Effects of Glyphosate-, Glufosinate- and Flazasulfuron-Based Herbicides on Soil Microorganisms in a Vineyard.

In a vineyard we examined the effects of broad-spectrum herbicides with three different active ingredients (glyphosate, glufosinate, flazasulfuron) on soil microorganisms. Mechanical weeding served as control treatment. Treatments were applied within grapevine rows and soil samples taken from there in 10-20 cm depth 77 days after application. Fungi were analyzed using classical sequencing technology and bacteria using next-generation sequencing. The number of colony-forming units (CFU) comprising bacteria, yeasts and molds was higher under flazasulfuron compared to all other treatments which had similar CFU levels. Abundance of the fungus Mucor was higher under flazasulfuron than glufosinate and mechanical weeding; Mucor was absent under glyphosate. Several other fungi taxa were exclusively found under a specific treatment. Up to 160 different bacteria species were found - some of them for the first time in vineyard soils. Total bacterial counts under herbicides were on average 260% higher than under mechanical weeding; however due to high variability this was not statistically significant. We suggest that herbicide-induced alterations of soil microorganisms could have knock-on effects on other parts of the grapevine system.

Four-trophic level food webs reveal the cascading impacts of an invasive plant targeted for biocontrol.

Biological invasions are a major threat to biodiversity and as such understanding their impacts is a research priority. Ecological networks provide a valuable tool to explore such impacts at the community level, and can be particularly insightful for planning and monitoring biocontrol programmes, including the potential for their seldom evaluated indirect non-target effects. Acacia longifolia is among the worst invasive species in Portugal, and has been recently targeted for biocontrol by a highly specific gall-wasp. Here we use an ambitious replicated network approach to: (1) identify the mechanisms by which direct and indirect impacts of A. longifolia can cascade from plants to higher trophic levels, including gallers, their parasitoids and inquilines; (2) reveal the structure of the interaction networks between plants, gallers, parasitoids and inquilines before the biocontrol; and (3) explore the potential for indirect interactions among gallers, including those established with the biocontrol agent, via apparent competition. Over a 15-month period, we collected 31,737 galls from native plants and identified all emerging insects, quantifying the interactions between 219 plant-, 49 galler-, 65 parasitoid- and 87 inquiline-species-one of the largest ecological networks to date. No galls were found on any of the 16 alien plant species. Invasion by A. longifolia caused an alarming simplification of plant communities, with cascading effects to higher trophic levels, namely: a decline of overall gall biomass, and on the richness, abundance and biomass of galler insects, their parasitoids, and inquilines. Correspondingly, we detected a significant decline in the richness of interactions between plants and galls. The invasion tended to increase overall interaction evenness by promoting the local extinction of the native plants that sustained more gall species. However, highly idiosyncratic responses hindered the detection of further consistent changes in network topology. Predictions of indirect effects of the biocontrol on native gallers via apparent competition ranged from negligible to highly significant. Such scenarios are incredibly hard to predict, but even if there are risks of indirect effects it is critical to weigh them carefully against the consequences of inaction and invasive species spread.

Review: biosafety assessment of Bt rice and other Bt crops using spiders as example for non-target arthropods in China.

Since the birth of transgenic crops expressing Bacillus thuringiensis (Bt) toxin for pest control, the public debate regarding ecological and environmental risks as well as benefits of Bt crops has continued unabated. The impact of Bt crops, especially on non-target invertebrates, has received particular attention. In this review, we summarize and analyze evidences for non-target effects of Bt rice on spiders, major predators in rice fields. Bt rice has been genetically modified to express the Bt protein, which has been shown to be transferred and accumulate in spiders as part of their food chain. Moreover, the Bt protein exhibits unintended effects on the physiology of spiders and spreads to higher trophic levels. Spiders possess unique physiological and ecological characteristics, revealing traits of surrogate species, and are thus considered to be excellent non-target arthropod model systems for study of Bt protein impacts. Due to the complexities of Bt protein transfer and accumulation mechanisms, as well as the apparent lack of information about resulting physiological, biochemical, and ecological effects on spiders, we raise questions and provide recommendations for promising further research.

Antifungal genes expressed in transgenic pea (Pisum sativum L.) do not affect root colonization of arbuscular mycorrhizae fungi.

Genetically modified crops have raised concerns about unintended consequences on non-target organisms including beneficial soil associates. Pea transformed with four antifungal genes 1-3 ß glucanase, endochitinase, polygalacturonase-inhibiting proteins, and stilbene synthase is currently under field-testing for efficacy against fungal diseases in Canada. Transgenes had lower expression in the roots than leaves in greenhouse experiment. To determine the impact of disease-tolerant pea or gene products on colonization by non-target arbuscular mycorrhizae and nodulation by rhizobium, a field trial was established. Transgene insertion, as single gene or stacked genes, did not alter root colonization by arbuscular mycorrhiza fungus (AMF) or root nodulation by rhizobium inoculation in the field. We found no effect of transgenes on the plant growth and performance although, having a dual inoculant with both AMF and rhizobium yielded higher fresh weight shoot-to-root ratio in all the lines tested. This initial risk assessment of transgenic peas expressing antifungal genes showed no deleterious effect on non-target organisms.

Quality of laboratory studies assessing effects of Bt-proteins on non-target organisms: minimal criteria for acceptability.

The potential risks that genetically modified plants may pose to non-target organisms and the ecosystem services they contribute to are assessed as part of pre-market risk assessments. This paper reviews the early tier studies testing the hypothesis whether exposure to plant-produced Cry34/35Ab1 proteins as a result of cultivation of maize 59122 is harmful to valued non-target organisms, in particular Arthropoda and Annelida. The available studies were assessed for their scientific quality by considering a set of criteria determining their relevance and reliability. As a case-study, this exercise revealed that when not all quality criteria are met, weighing the robustness of the study and its relevance for risk assessment is not obvious. Applying a worst-case expected environmental concentration of bioactive toxins equivalent to that present in the transgenic crop, confirming exposure of the test species to the test substance, and the use of a negative control were identified as minimum criteria to be met to guarantee sufficiently reliable data. This exercise stresses the importance of conducting studies meeting certain quality standards as this minimises the probability of erroneous or inconclusive results and increases confidence in the results and adds certainty to the conclusions drawn.

Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review.

Mosquitoes (Diptera: Culicidae) are a key threat for millions of people worldwide, since they act as vectors for devastating parasites and pathogens. Mosquito young instars are usually targeted with organophosphates, insect growth regulators and microbial control agents. Indoors residual spraying and insecticide-treated bed nets are also employed. However, these chemicals have strong negative effects on human health and the environment. Newer and safer tools have been recently implemented to enhance control of mosquitoes. In this review, I focus on characterization, effectiveness, and non-target effects of mosquitocidal nanoparticles synthesized using botanical products (mosquitocidal nanoparticles, MNP). The majority of plant-fabricated MNP are silver ones. The synthesis of MNP is usually confirmed by UV-visualization spectroscopy, followed by scanning electron microscopy or transmission electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction studies. Interestingly, plant-synthesized metal nanoparticles have been reported as effective ovicides, larvicides, pupicides, adulticides, and oviposition deterrents against different mosquito species of medical and veterinary importance. Few parts per million of different MNP are highly toxic against the malaria vector Anopheles stephensi, the dengue vector Aedes aegypti, and the filariasis mosquito Culex quiquefasciatus. However, despite the growing number of evidences about the effectiveness of MNP, moderate efforts have been carried out to shed light on their possible non-target effects against mosquito's natural enemies and other aquatic organisms. In the final section, particular attention was dedicated to this issue. A number of hot areas that need further research and cooperation among parasitologists and entomologists are highlighted.

α-Humulene and ß-elemene from Syzygium zeylanicum (Myrtaceae) essential oil: highly effective and eco-friendly larvicides against Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus (Diptera: Culicidae).

Mosquitoes transmit serious pathogens and parasites to humans and animals, including malaria, dengue, Japanese encephalitis and filariasis. The extensive use of chemical pesticides leads to the development of resistance in mosquito vector populations and serious non-target effects on human health and the environment. Myrtaceae plants can be a useful reservoir of natural products effective against Culicidae young instars. In this research, we evaluated the mosquitocidal potential of the essential oil (EO) from Syzygium zeylanicum leaves against larvae of three mosquitoes of medical and veterinary importance, the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus, and the Japanese encephalitis vector Culex tritaeniorhynchus. The chemical composition of the EO was analyzed by gas chromatography-mass spectroscopy. GC-MS revealed that the S. zeylanicum EO contained at least 18 compounds. The major chemical components were α-humulene (37.8.5 %) and ß-elemene (10.7 %). The EO had a significant toxic effect against early third-stage larvae of An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus, with LC50 values of 83.11, 90.45, and 97.96 µg/ml, respectively. The two major constituents extracted from the S. zeylanicum EO were tested individually for acute toxicity against larvae of the three mosquito vectors. α-Humulene and ß-elemene appeared highly effective against An. subpictus (LC50 = 6.19 and 10.26 µg/ml, respectively), followed by Ae. albopictus (LC50 = 6.86 and 11.15 µg/ml) and Cx. tritaeniorhynchus (LC50 = 7.39 and 12.05 µg/ml). Furthermore, the EO and its major components was safe towards the non-target fish Gambusia affinis; LC50 values were 20374.26, 1024.95, and 2073.18 µg/ml, respectively for EO, α-humulene and ß-elemene. Overall, this study highlighted that the acute toxicity of S. zeylanicum EO towards mosquito larvae was mainly due to the presence of α-humulene and ß-elemene. Furthermore, we pointed out the concrete possibility to exploit these two compounds from S. zeylanicum EO as highly effective larvicides against young instars of An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus, with little effect on non-target organisms.

Fabrication of nano-mosquitocides using chitosan from crab shells: Impact on non-target organisms in the aquatic environment.

Mosquitoes are arthropods of huge medical and veterinary relevance, since they vector pathogens and parasites of public health importance, including malaria, dengue and Zika virus. Currently, nanotechnology is considered a potential eco-friendly approach in mosquito control research. We proposed a novel method of biofabrication of silver nanoparticles (AgNP) using chitosan (Ch) from crab shells. Ch-AgNP nanocomposite was characterized by UV-vis spectroscopy, FTIR, SEM, EDX and XRD. Ch-AgNP were tested against larvae and pupae of the malaria vector Anopheles stephensi obtaining LC50 ranging from 3.18 ppm (I) to 6.54 ppm (pupae). The antibacterial properties of Ch-AgNP were proved against Bacillus subtilis, Klebsiella pneumoniae and Salmonella typhi, while no growth inhibition was reported in assays conducted on Proteus vulgaris. Concerning non-target effects, in standard laboratory considtions the predation efficiency of Danio rerio zebrafishes was 68.8% and 61.6% against I and II instar larvae of A. stephensi, respectively. In a Ch-AgNP-contaminated environment, fish predation was boosted to 89.5% and 77.3%, respectively. Quantitative analysis of antioxidant enzymes SOD, CAT and LPO from hepatopancreas of fresh water crabs Paratelphusa hydrodromous exposed for 16 days to a Ch-AgNP-contaminated aquatic environment were conducted. Notably, deleterious effects of Ch-AgNP contaminating aquatic enviroment on the non-target crab P. hydrodromous were observed, particularly when doses higher than 8-10ppm are tested. Overall, this research highlights the potential of Ch-AGNP for the development of newer control tools against young instar populations of malaria mosquitoes, also highlighting some risks concerned the employ of nanoparticles in aquatic environments.

Eco-friendly larvicides from Indian plants: Effectiveness of lavandulyl acetate and bicyclogermacrene on malaria, dengue and Japanese encephalitis mosquito vectors.

Mosquitoes (Diptera: Culicidae) are a key threat for millions of people and animals worldwide, since they act as vectors for devastating pathogens and parasites, including malaria, dengue, Japanese encephalitis, filiariasis and Zika virus. Mosquito young instars are usually targeted using organophosphates, insect growth regulators and microbial agents. Indoor residual spraying and insecticide-treated bed nets are also employed. However, these chemicals have negative effects on human health and the environment and induce resistance in a number of vectors. In this scenario, newer and safer tools have been recently implemented to enhance mosquito control. The concrete potential of screening plant species as sources of metabolites for entomological and parasitological purposes is worthy of attention, as recently elucidated by the Y. Tu's example. Here we investigated the toxicity of Heracleum sprengelianum (Apiaceae) leaf essential oil and its major compounds toward third instar larvae of the malaria vector Anopheles subpictus, the arbovirus vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. GC-MS analysis showed that EO major components were lavandulyl acetate (17.8%) and bicyclogermacrene (12.9%). The EO was toxic to A. subpictus, A. albopictus, and C. tritaeniorhynchus, with LC50 of 33.4, 37.5 and 40.9µg/ml, respectively. Lavandulyl acetate was more toxic to mosquito larvae if compared to bicyclogermacrene. Their LC50 were 4.17 and 10.3µg/ml for A. subpictus, 4.60 and 11.1µg/ml for A. albopictus, 5.11 and 12.5µg/ml for C. tritaeniorhynchus. Notably, the EO and its major compounds were safer to three non-target mosquito predators, Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC50 ranging from 206 to 4219µg/ml. Overall, this study highlights that H. sprengelianum EO is a promising source of eco-friendly larvicides against three important mosquito vectors with moderate toxicity against non-target aquatic organisms.