Bio‐safety evaluation of Cry1Ac,Cry2Ab,Cry1Ca,Cry1F and Vip3Aa on Harmonia axyridis larvae

Dietary exposure studies are initial steps in environmental risk assessments of genetically engineered plants on non‐target organisms. These studies are conducted in the laboratory where surrogate species are exposed to purified and biologically active insecticidal compounds at higher concentrations than those expected to occur in transgenic crops foliage. Thus, dietary exposure (early tier) tests provide robust data needed to make general conclusions about the susceptibility of the surrogate species to the test substance. For this, we developed suitable artificial diet and used it to establish a dietary exposure test for assessing the toxicity of midgut‐active insecticidal compounds to the larvae of the Asian ladybird beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Using boric acid as a model compound, we validated the bioassay established for H. axyridis larvae. An artificial diet containing boric acid which negatively affected survival, development and adult weights was offered to larvae and indicated that the bioassay was able to detect toxic effects of insecticidal substances incorporated in diets. Using this dietary exposure test, environmental risk assessment of Cry1Ac, Cry2Ab, Cry1Ca, Cry1F and the non‐Cry protein Vip3Aa was evaluated by analysing pupation rates, adult emergence rates, 7‐day larval weights, and freshly emerged male and female weights among the toxin treatments and a pure artificial diet. These life‐table parameters did not vary among artificial diets containing 200 μg/g Bt proteins or pure artificial diet. In contrast, boric acid adversely affected all life‐table parameters. Thus on these bases, we concluded H. axyridis larvae are not sensitive to these Bt proteins expressed in genetically engineered crops.     

Synergy between fipronil and amitraz in a <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> tick residual contact test

Amitraz, a formamidine acaricide, has been reported to act as a synergist of contact insecticides and acaricides in both agriculture and animal health. A laboratory contact test was therefore conducted to determine whether amitraz at 12.5 ppm could improve the contact potency of fipronil on ticks. A controlled glass vial bioassay was used to assess the efficacy of fipronil alone, amitraz alone, and fipronil plus amitraz on unfed adult Rhipicephalus sanguineus. Assessments of lethality were made at 6, 24, and 48 h after the introduction of the ticks to the vials. No significant mortality was observed in the control treatment or in the amitraz alone treatment. Concentration and time dependent mortality rates were observed in ticks exposed to fipronil alone or fipronil plus amitraz, with higher mortality observed in the latter group. Results from this study gave synergistic EC50 ratios between fipronil alone and fipronil plus amitraz of >7.3, 137 and 97 at 6, 24, and 48 h, respectively. A similar response was seen at the EC90 level. These results indicate that fipronil was synergized by amitraz in this adult tick residual contact study. The addition of amitraz to fipronil also provided a significant improvement in the speed of kill.     

Antiperoxidative and Antiinflammatory Effect of <Emphasis Type="Italic">Sida Cordifolia</Emphasis> Linn. on Quinolinic Acid Induced Neurotoxicity

Sida cordifolia is a plant belonging to the Malvaceae family used in many ayurvedic preparations. This study aimed at assessing the effects of ethanolic extract of Sida cordifolia root on quinolinic acid (QUIN) induced neurotoxicity and to compare its effect with the standard drug deprenyl in rat brain. Rats were divided into six groups: (1) control group (2) QUIN (55 μg/100 g bwt/day) (3) 50% ethanolic plant extract treated group (50 mg/100 g bwt/day) (4) Deprenyl (100 μg/100 g bwt/day) (5) QUIN (55 μg/100 g bwt/day) + 50% ethanolic plant extract treated group (50 mg/100 g bwt/day) (6) QUIN (55 μg/100 g bwt/day) + Deprenyl (100 μg/100 g bwt/day). At the end of the experimental period a status of lipid peroxidation products, protein peroxidation product, activities of the scavenging enzymes and the activities of the inflammatory markers were analyzed. Results revealed that the lipid peroxidation products decreased and the activities of the scavenging enzymes increased significantly in the brain of the plant extract treated group, deprenyl treated group and also in the coadminstered groups. The activities of markers of inflammatory responses such as cyclooxygenase and lipoxygenase were found to be significantly increased in the QUIN treated rats and this was decreased upon the administration of plant extract and deprenyl. In short, the study revealed that 50% ethanolic extract of Sida cordifolia has got potent antioxidant and antiinflammatory activity and the activity is comparable with the standard drug deprenyl.     

Oral toxicity of abamectin, boric acid, fipronil, and hydramethylnon to laboratory colonies of Argentine ants (Hymenoptera: Formicidae)

Comprehensive laboratory baiting studies with the 4 insecticides abamectin, boric acid, fipronil, and hydramethylnon were conducted against Argentine ant, Linepithema humile (Mayr). Serial dilutions of each insecticide prepared in a 25% sucrose solution were provided for 24 h or 14 d to groups of workers and queens. Sucrose-water baits containing 1 x 10(-5)% fipronil and 0.1% hydramethylnon provided complete mortality of L. humile workers baited for 24 h. Baits containing 1 x 10(-4) and 1 x 10(-5)% fipronil provided 100% kill of queens. In the continuous baiting, 0.5% boric acid, 1 x 10(-6)% fipronil, and 0.025% hydramethylnon provided 100% mortality of workers. The queens and workers provided baits containing 1 x 10(-5)% fipronil and 0.5% boric acid all died within 14 d. Low concentrations of fipronil and boric acid in sucrose may be promising candidates for Argentine ant control.     

Sublethal Effect of Copper Toxicity Against Histopathological Changes in the Spiny Lobster, <Emphasis Type="Italic">Panulirus homarus</Emphasis> (Linnaeus, 1758)

The tissue damage induced by various organic pollutants in aquatic animals is well documented, but there is a dearth of information relating to the histological alterations induced by copper in the spiny lobster. In the present study, intermoult juveniles of the spiny lobster Panulirus homarus (average weight 150–200 g) were exposed to two sublethal concentrations of the copper (9.55 and 19.1 μg/l) for a period of 28 days. The muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of the lobsters were then dissected out and processed for light microscopic studies. Exposure to copper was found to result in several alterations in the histoarchitecture of the muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of P. homarus. The alterations included disruption and congestion of muscle bundle in muscle tissue; blackened haemocytes; distended lumen and F cell; necrosis of the tubules of the hepatopancreas; disarrangement of circular muscle of the midgut; accumulation of haemocytes in the haemocoelic space; swelling and fusion of lamellae; abnormal gill tips; hyperplastic, necrotic, and blackened secondary gill lamellae of the gills; damaged neurosecretory cell and sensory and motor fibre; necrotic of the thoracic ganglion; dispersedly arranged muscle bands; clumped satellite cells and nucleus of the heart. The results obtained suggest that the muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of lobsters exposed to copper were structurally altered. Such alterations could affect vital physiological functions, such as absorption, storage and secretion of the hepatopancreas, digestion of gut and respiration, osmotic and ionic regulations of the gills, which in turn could ultimately affect the survival and growth of P. homarus. Thus, all possible remedial measures should be adopted to prevent the occurrence of copper contamination in the aquatic environment.     

<Emphasis Type="Italic">Toxoneuron nigriceps</Emphasis> parasitization delays midgut replacement in fifth-instar <Emphasis Type="Italic">Heliothis virescens</Emphasis> larvae

We have analyzed the effects of Toxoneuron nigriceps parasitization on the midgut development of its host Heliothis virescens. In parasitized H. virescens larvae, the midgut epithelium undergoes a complete replacement, which is qualitatively not different to that observed in synchronous unparasitized larvae, with similar temporal profiles of cell death and metabolic activity. However, the whole gut replacement process is significantly delayed in parasitized larvae, with complete differentiation of the new gut epithelium being observed 4 days later than in unparasitized controls. The administration of juvenile hormone before commitment and of 20-hydroxyecdysone (20E) after commitment delays and fosters, respectively, the replacement process of the midgut epithelium; moreover, the injection of 20E into developmentally arrested and 20E-deficient host last-instar larvae parasitized by T. nigriceps immediately triggers regular gut development. These hormone-based experiments suggest that endocrine alterations in the larval host, induced by T. nigriceps parasitism, are responsible for the temporal alterations in the gut replacement process. The role of this parasitoid-induced developmental change in the host regulation process is discussed. This work was partially supported by FAR 2006–2007 (University of Insubria) to G.T., by MIUR-FIRB-COFIN (grant no. RBNE01YXA8/2004077251), and by the Centro Grandi Attrezzature (University of Insubria).     

羧酸酯酶介导的小菜蛾对氟虫腈的抗性

【目的】羧酸酯酶（carboxylesterases, CarEs）是昆虫重要的解毒代谢酶之一,可以介导靶标昆虫对多种杀虫剂的代谢抗性。本研究检测了羧酸酯酶对小菜蛾Plutella xylostella 抗药性的介导功能,旨在阐明羧酸酯酶在小菜蛾代谢解毒中的生理生化和分子机理。【方法】采用点滴法测定氟虫腈对小菜蛾敏感种群和抗氟虫腈种群的毒力,以及羧酸酯酶抑制剂磷酸三苯酯（triphenyl phosphate, TPP）对氟虫腈的增效作用;以LC₃₀和LC₅₀浓度的氟虫腈处理抗性小菜蛾,测定药剂处理后CarEs酶活性的变化;利用qRT-PCR技术分析Pxae22和Pxae31两个基因在小菜蛾不同发育阶段、组织和种群的表达模式;利用dsRNA干扰Pxae22和Pxae31后观察基因的表达变化和小菜蛾3龄幼虫对药剂敏感性的变化。【结果】TPP可以削弱小菜蛾3龄幼虫对氟虫腈的抗性,增效倍数约为6倍;使用较低剂量（LC₃₀和LC₅₀）氟虫腈处理小菜蛾3龄幼虫后,处理组CarEs比活力明显高于对照,提示氟虫腈对小菜蛾CarEs活性具有诱导作用。对羧酸酯酶基因Pxae22和Pxae31在小菜蛾不同发育阶段、4龄幼虫不同组织和不同种群3龄幼虫中的表达模式分析发现,这两个基因在小菜蛾4龄幼虫中的表达量最高;在4龄幼虫中以中肠组织中的表达量较高,头、表皮、脂肪体中的表达量很低; 抗性种群中的表达量显著高于敏感种群。通过干扰 Pxae22和 Pxae31后的qRT-PCR验证,两个基因的表达量均显著降低,进一步的氟虫腈毒力测定发现,干扰P xae22和 Pxae31后的小菜蛾3龄幼虫对氟虫腈的敏感性分别增加了1.63倍和1.73倍。【结论】羧酸酯酶在小菜蛾对氟虫腈解毒代谢中具有重要作用;Pxae22和Pxae31是小菜蛾的两个抗性相关基因,其表达水平的变化直接影响小菜蛾对氟虫腈的敏感性。     

Antifouling and antibacterial compounds from a marine fungus <Emphasis Type="Italic">Cladosporium</Emphasis> sp. F14

To investigate the antifouling secondary metabolites from marine-derived fungi, we used bioassay-guided column chromatography techniques, such as HPLC, to separate and purify compounds from Cladosporium sp. F14. Extensive spectral analyses including 1D NMR spectra and MS were employed for structure elucidation of the compounds. Antilarval activity of the compounds was evaluated in settlement inhibition assays with laboratory-reared Balanus amphitrite and Bugula neritina larvae, while antibacterial activity was assessed with disc diffusion bioassay on growth inhibition of six marine bacterial species. In total, nine compounds were obtained. Among them, 3-phenyl-2-propenoic acid, cyclo-(Phe-Pro) and cyclo-(Val-Pro) had various antibacterial activities against three fouling bacteria, furthermore, 3-phenyl-2-propenoic acid and bis(2-ethylhexyl)phthalate effectively inhibited larval settlement of B. neritina and B. amphitrite larvae, respectively, indicating that the two compounds are potential natural antifouling agents.     

土荆芥精油预处理后小菜蛾幼虫对氟虫腈敏感度的变化

采用微量点滴法测定了土荆芥Chenopodium ambrosioides精油预处理前后氟虫腈对小菜蛾Plutella xylostella 2龄幼虫的触杀作用,采用生化分析方法研究了土荆芥精油预处理前后小菜蛾幼虫体内乙酰胆碱酯酶（AChE）、羧酸酯酶（CarE）和谷胱甘肽-S-转移酶（GSTs）的比活力。研究结果表明,1μL·mL^-1和10μL·mL^-1的土荆芥精油预处理可增加小菜蛾幼虫对氟虫腈的敏感度,LC50分别为氟虫腈单用时的0.66倍和0.44倍;当用1μL·mL^-1和10μL·mL^-1土荆芥精油预处理小菜蛾2龄幼虫2h后,再用氟虫腈致死中量点滴处理,小菜蛾存活幼虫体内AChE、CarE和GSTs比活力均低于氟虫腈单独处理组。研究结果说明土荆芥精油预处理对氟虫腈的增效作用可能与主要抗性相关酶系活性变化有关。     

Histopathology and ultrastructure of midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) fed Bt-cotton

The interaction of Cry toxins from Bacillus thuringiensis in the midgut of some insect larvae determines their efficacies as insecticides, due to the expression and availability of sites of action of the toxin in the midgut. Researches point out cases of resistance to Cry toxin due to alterations in the binding sites in columnar cell membrane. We analyzed the effects of Cry1Ac toxin expressed by Bt-cotton plants on Alabama argillacea midgut morphophysiology clarifying in levels of morphological and ultrastructural. Larvae in the 4th instar of A. argillacea after 20 min from ingesting Bt-cotton leaves expressing 0.183 ng of Cry1Ac exhibited ultrastructural and morphological modifications in the columnar cells with significant changes in the mitochondrial polymorphism, cytoplasmic vacuolization, microvillus and basal labyrinth. Expressive morphological alterations were also observed in the goblet cells indicating that the columnar cells are not the only target of the Cry1Ac toxin. The regenerative cells did not modify their structures and exhibited decrease in regeneration capacity. In conclusion, the ingestion of 0.183 ± 0.077 ng of Cry1Ac was enough to promote alterations in the columnar and goblet cells, besides reducing significantly the number of regenerative cells, which may have contributed to larval death. Nevertheless, further studies are necessary to determine the true cause of death.     

Environmentally relevant cadmium concentrations affect development and induce apoptosis of <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> larvae cultured in vitro

Sea urchin embryos and larvae represent suitable model systems on where to investigate the effects of heavy metals on development and cell viability. Here, we tested the toxic effects of low (10⁻¹² M), medium (10⁻⁹ M), and high (10⁻⁶ M) cadmium chloride concentrations, mimicking unpolluted, moderately and highly polluted seawaters, respectively, on Paracentrotus lividus sea urchins offspring. Larvae were continuously treated from fertilization and inspected at time intervals comprised between 10 and 30 days of development. Delays and/or morphological abnormalities were firstly evident in larvae treated for 15 days with high cadmium (10⁻⁶ M) and for 25 days with medium cadmium (10⁻⁹ M). Major defects consisted in the reduction and lack of arms and skeleton elongation. No obvious differences with respect to controls were observed in embryos/larvae exposed to low cadmium (10⁻¹² M), even after 30 days of exposure. Using in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay on larvae whole mounts, we detected apoptosis after 10 days of treatment with 10⁻⁶ and 10⁻⁹ M CdCl_2, when no morphological abnormalities were recognizable yet. Supernumerary apoptotic cells were found in arm buds, ciliary bands, and apex. In conclusion, echinoderm embryos and larvae represent candidates of choice for the study of stress and defense mechanisms activated by cadmium exposure.     

Topical Treatment of Dermatophytic Lesion on Mice (<Emphasis Type="Italic">Mus musculus</Emphasis>) Model

Antidermatophytic potential of three weed plants viz. Tridax procumbens L., Capparis decidua (forsk) Edgew and Lantana camara L. were explored and experimentally induced dermatophytic lesion was topically treated in mice. Microbroth dilution method was carried out for determination of MIC and MFC of different extracts of selected plants. In animal studies, mice were experimentally inoculated with Trichophyton mentagrophytes and infected animals were topically treated with 5 mg/g terbinafine and two concentrations, i.e., 5 and 10 mg/g of test extract ointment. Complete recovery from the infection was observed on 12th day of treatment for reference drug terbinafine (5 mg/g) and 10 mg/g concentration of test extract ointment whereas 5 mg/g concentration of test extract ointment showed complete cure on 16th day of treatment. Fungal burden was also calculated by culturing skin scrapings from infected animals of different groups. Test extract ointment successfully treated induced dermatophytosis in mice without any disease recurrence incidences, thereby indicating efficacy of test extract as an excellent topical antifungal agent for the cure of dermatophytosis.     

Interaction of Salivary and Midgut Proteins of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> with Soybean Trypsin Inhibitor

Feeding of Helicoverpa armigera larvae on semi-synthetic diet containing Soybean trypsin inhibitor (STI) resulted in disappearance of STI sensitive protease in salivary and midgut protease extract. This might be due to in situ inhibition by dietary STI. STI was largely degraded within 1 h of incubation with total salivary protease (1:1). Degradation was relatively low in midgut proteases. STI interacting proteins were isolated from saliva and midgut extracts of larvae fed on STI supplemented diet using affinity column. Most of the isolated proteins showed caseinolytic activity in zymogram. Denovo sequencing data of seven different peptides selected from trypsin digested total protein showed similarity to chymotrypsinogen, serine protease, aminopeptidase N, peroxidase, hypothetical protein and muscle specific protein.     

Efficacy of Boric Acid,Monopotassium Phosphate and Sodium Metabisulfite on the Control of Apple Scab

In vitro experiments indicated that boric acid, monopotassium phosphate, sodium metabisulfite and synthetic fungicide fluopyram + tebuconazole were effective in inhibiting conidia germination and germ‐tube elongation of Venturia inaequalis. Monopotassium phosphate even at the highest concentration used in the study reduced conidia germination and germ‐tube elongation of V. inaequalis by 22.1% and 28.8%, respectively; however, the difference between two compounds at lower concentrations except 0.05% (for conidia germination) and 0.1% (for germ‐tube elongation) of boric acid was statistically significant (P ≤ 0.05). Complete inhibition was achieved by 0.01% sodium metabisulfite, 0.035% fluopyram + tebuconazole and 0.2% boric acid. Two orchard trials were conducted on the highly susceptible cv. Mutsu to apple scab to ascertain the efficacy of 0.2% boric acid, 0.5% monopotassium phosphate, 0.5% sodium metabisulfite and 0.035% fluopyram + tebuconazole for the control of apple scab. In both 2013 and 2014, except for the applications of monopotassium phosphate and sodium metabisulfite, the applications of boric acid and fluopyram + tebuconazole to trees at 10‐day intervals significantly reduced disease incidence and severity on leaves and fruit compared to the water‐treated control. In both years, the efficacy of boric acid and fluopyram + tebuconazole treatments was similar in reducing both disease incidence and severity on leaves and fruit in all monthly assessments from July to September. All treatments were neither phytotoxic to leaves and fruit nor did they adversely affect quality parameters of harvested fruit. These results show that boric acid treatment may be applied as an alternative chemical for the control of apple scab.     

Insecticide-tolerant and plant growth promoting <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. (<Emphasis Type="Italic">vigna</Emphasis>) improves the growth and yield of greengram [<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek] in insecticide-stressed soils

This study was designed to identify rhizobial strains specific to greengram expressing higher tolerance against insecticides, fipronil and pyriproxyfen, and synthesizing plant growth regulators even amid insecticide-stress. Of the 50 bradyrhizobial isolates, the Bradyrhizobium sp. strain MRM6 showed tolerance up to 1,600 μg mL⁻¹ against each of fipronil and pyriproxyfen. The tolerant Bradyrhizobium sp. (vigna) produced plant growth promoting substances in substantial amounts, both in the presence and absence of insecticides. The strain MRM6 was further used to investigate its impact on greengram grown in soils treated with 200 (the recommended dose), 400 and 600 μg kg⁻¹ soil of fipronil and 1,300 (the recommended dose), 2,600 and 3,900 μg kg⁻¹ soil of pyriproxyfen. Fipronil at 600 μg kg⁻¹ soils and pyriproxyfen at 3,900 μg kg⁻¹ soils had greatest toxic effects and decreased plant biomass, symbiotic efficiency, nutrient uptake and seed yield of greengram plants. The Bradyrhizobium sp. (vigna) inoculant when used with fipronil and pyriproxyfen significantly increased the measured parameters compared to the plants grown in soils treated solely with the same concentration of each insecticide. This study inferred that the Bradyrhizobium sp. (vigna) strain MRM6 may be exploited as bio-inoculant to increase the productivity of greengram exposed to insecticide-stressed soils.     

Toxin-binding proteins isolated from yellow mealworm <Emphasis Type="Italic">Tenebrio molitor</Emphasis> and wax moth <Emphasis Type="Italic">Galleria mellonella</Emphasis>

A 67-kDa protein that can specifically bind the activated Cry9A endotoxin under ligand-blotting conditions was purified from midgut epithelium apical membranes of wax moth Galleria mellonella by affinity chromatography. N-Terminal amino acid sequencing enabled identification of this protein as aminopeptidase N. In similar experiments, 66- and 58-kDa proteins specific to endotoxin Cry3A were isolated from the midgut epithelium apical membranes of Tenebrio molitor larvae. Mass spectrometry showed close similarity of the 58-kDa protein to the Tenebrio molitor α-amylase.     

Growth and morphological development of laboratory-reared larval and juvenile three-spot gourami <Emphasis Type="Italic">Trichogaster trichopterus</Emphasis>

Morphological development, including the body proportions, fins, pigmentation and labyrinth organ, in laboratory-hatched larval and juvenile three-spot gourami Trichogaster trichopterus was described. In addition, some wild larval and juvenile specimens were observed for comparison. Body lengths of larvae and juveniles were 2.5 ± 0.1 mm just after hatching (day 0) and 9.2 ± 1.4 mm on day 22, reaching 20.4 ± 5.0 mm on day 40. Aggregate fin ray numbers attained their full complements in juveniles >11.9 mm BL. Preflexion larvae started feeding on day 3 following upper and lower jaw formation, the yolk being completely absorbed by day 11. Subsequently, oblong conical teeth appeared in postflexion larvae >6.4 mm BL (day 13). Melanophores on the body increased with growth, and a large spot started forming at the caudal margin of the body in flexion postlarvae >6.7 mm BL, followed by a second large spot positioned posteriorly on the midline in postflexion larvae >8.6 mm BL. The labyrinth organ differentiated in postflexion larvae >7.9 mm BL (day 19). For eye diameter and the first soft fin ray of pelvic fin length, the proportions in laboratory-reared specimens were smaller than those in wild specimens in 18.5–24.5 mm BL. The pigmentation pattern of laboratory-reared fish did not distinctively differ from that in the wild ones. Comparisons with larval and juvenile morphology of a congener T. pectoralis revealed several distinct differences, particularly in the numbers of myomeres, pigmentations and the proportional length of the first soft fin ray of the pelvic fin.     

Effects of coadministration of chemical insecticides with nucleopolyhedrovirus SpltNPV on the dietary intake of the common cutworm <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Lepidoptera: Noctuidae)

Chemical insecticides were administered to larvae of the common cutworm Spodoptera litura (Fabricius) concomitantly with a viral insecticide containing nucleopolyhedrovirus SpltNPV, in order to decrease the level of dietary intake of the virus-infected larvae. Aqueous solutions of imidacloprid (Admire), flubendiamide (Phenix), or cartap (Padan) at non-lethal doses, together with a lethal dose of SpltNPV occlusion bodies (Hasumon-Tenteki), were prepared. The surfaces of small blocks of food were treated with the mixtures and presented to S. litura larvae for 24 h. On the following day, a new diet block was provided which had been treated with one-third or one-fourth of the initial dose of the chemical insecticide. The larvae were reared for 5 days and the level of dietary intake was recorded. The administration of flubendiamide and cartap decreased the dietary intake more strongly in virus-infected larvae than in non-infected larvae.     

Influence supplementary feeding on bark browsing by red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) under experimental conditions

The effect of various supplementary feeds on bark browsing was studied in red deer under controlled conditions. Fifteen female red deer were randomly assigned to five dietary treatments (n = 3 animals each): meadow hay as a control (C), combinations of meadow hay with grass silage (GS), maize silage (MS), maize silage with oats (MSO) or a pelleted concentrate (PC). The deer were kept in enclosures where we offered fresh spruce stems daily. The experiment consisted of two periods. In the first period (A), the animals had free access to food during 24 h, while in the second period (B), the access was restricted to night hours only. In both periods, the supplementary feeds were available ad libitum. Mean daily consumption of browsed bark in the control group in period A was 1,053 g while in the remaining groups it ranged from six to 20 g. In period B, simulating the conditions in disturbed hunting areas, bark consumption in all groups increased considerably, the highest increase being found in groups GS (430 vs. 19 g/day) and MS (101 vs. 6 g/day). In period A, the highest dry matter (DM) intake including browse was found in group MS (2,816 g/day) and the lowest one in group GS (2,307 g/day). In period B, the highest (2,763 g/day) and the lowest (2,153 g/day) DM intake was observed in groups PC and MSO, respectively. If red deer is fed supplementary, meadow hay should be combined with maize silage, grass silage or concentrate to reduce bark browsing in forests. The bark browsing rate may increase considerably if red deer are disturbed or their grazing cycle is impaired.     

Histological changes in the Dengue vector,Aedes aegypti (Diptera: Culicidae) larvae treated with neem oil loaded niosomes

Mosquitoes are one of the greatest threats to human health around the globe. They act as vectors for common diseases like Malaria, Dengue, Chikungunya, etc. Niosomes encapsulated with neem oil showed a significant mortality rate against Aedes aegypti larvae when treated for 24 h. In this study, the histological changes that led to the mortality of the Aedes aegypti mosquito larvae were studied. Organs of the late III-instar stage larvae such as head, optic lobes, cuticle, adipose tissue, midgut region, haemolymph were investigated. Several histological alterations such as disorientation of the brain and antenna in the head part, damage in the optic lobe and microvilli were observed. Total disruption was seen in the inner and outer retractor muscles of the larval body. The midgut and hindgut regions were disintegrated due to the damage to the fat bodies in the region. A Series of such histological changes in the body of mosquito larvae compared to the control larvae hindered metabolic functions leading to death. The results suggested that the neem oil loaded niosomes could be used as a biocontrol agent against the Dengue vector, Aedes aegypti larvae.