Antimicrobial activity and acute and chronic toxicity of the essential oil of Lippia origanoides

Currently, there is a growing interest in medicinal plants, because of an increased demand for alternate therapies. In this study, the antimicrobial activity and toxicity of the essential oil of Lippia origanoides (L. origanoides) were investigated. The essential oil of L. origanoides was extracted by steam-dragging distillation and its constituents were identified by chromatography coupled with mass spectrometry. Among the 15 compounds identified, the most abundant were carvacrol (29.00%), o-cymene (25.57%), and thymol methyl ether (11.50%). The essential oil was studied in antimicrobial assays to determine the MIC and MBC. The results indicated that a concentration of 120μL/mL of oil was sufficient to inhibit the growth of the following microorganisms: Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Salmonella cholerasuis (ATCC 10708). Acute and chronic toxic effects of orally administered oil were investigated in Wistar rats by using standard methods. Doses of 30, 60 and 120mg/kg of the essential oil did not induce significant changes in weight, behavior or hematological and biochemical parameters in the animals. There were no signs of any histopathological changes to the liver, kidneys or heart of the treated rats, suggesting that Lippia origanoides oil is non-toxic after oral administration in acute or chronic toxicity studies. The results obtained in this study show that the essential oil of L. origanoides has a high safety margin, with no detectable toxic effects in rats treated with doses to 120mg/kg. In addition, L. origanoides oil demonstrated potent antimicrobial activity against S. aureus, E. coli and S. cholerasuis. Based on these findings, this essential oil may have practical application as a veterinary antimicrobial.

Anti-predator adaptations in aquatic environments

Predation is an important selective force that has led to the evolution of a variety of fascinating anti-predator adaptations, such as many types of protective coloration and prey behaviours. Because the evolution of life has begun in the aquatic environment and many anti-predator adaptations are found already in relative primitive taxa, it is likely that many of these adaptations evolved initially in the aquatic environment. Yet, there has been surprisingly little research on the mechanisms and function of antipredator adaptations in aquatic systems. To understand the function of anti-predator adaptations and natural selection imposed on prey appearance and behaviour, I have investigated how protective coloration can be used, either as such or together with behavioural adaptations, to manipulate predator behaviour and decrease predation risk. To this end I conducted a series of behaviour ecological laboratory experiments in which I manipulated the visual appearance of artificial backgrounds and prey items. In paper I of this thesis, I investigated background choice as an anti-predator strategy, by observing the habitat choice of the least killifish (Heterandria formosa) between pairs of artificial backgrounds, both in the presence and absence of predation threat. It has been suggested that prey could decrease their risk of being detected by predators either by preferring backgrounds into which they blend or by preferring visually complex backgrounds. The least killifish preferred a background that matched their patterning to a background that mismatched it, showing that they are able to respond to cues of visual similarity between their colour pattern and the surrounding environment. Interestingly however, in female least killifish visual complexity of the background was a more important cue for habitat safety and may override or act together with background matching when searching for a safe habitat. It is possible that in females, preference for visually complex backgrounds is associated with lower opportunity costs than preference for matching backgrounds would be. Generally, the least killifish showed stronger preference while under predation threat, indicating that their background choice behaviour is an antipredator adaptation. Many aquatic prey species have eyespots, which are colour patterns that consist of roughly concentric rings and have received their name because they for humans often resemble the vertebrate eye. I investigated the anti-predator function of eyespots against predation by fish in papers II, III and IV. Some eyespots have been suggested to benefit prey by diverting the strikes of predators away from vital parts of the prey body or towards a direction that facilitates prey escape. Although proposed over a century ago, the divertive effect of eyespots has proven to be difficult to show experimentally. In papers II and III, I tested for divertive effect of eyespots towards attacking fish by presenting artificial prey with eyespots to laboratory reared three-spined sticklebacks (Gasterosteus aculeatus). I found that eyespots strongly influenced the behaviour of attacking sticklebacks and effectively drew their strikes towards the eyespots. To further investigate this divertive effect and whether the specific shape of eyespots is important for it, I tested in paper III the response of fish also to other markings than eyespots. I found that eyespots were generally more effective in diverting the first strikes of attacking fish compared to other prey markings. My findings suggest that the common occurrence of eyespots in aquatic prey species can at least partly be explained by the divertive effect of the eyespot shape, possibly together with the relative simple developmental mechanisms underlying circular colour patterns. An eyebar is a stripe that runs through the eye, and this pattern has been suggested to obscure the real eyes of the prey by visually blending parts of the eyes and head of the prey and by creating false edges. In paper III, I show that an eyebar effectively disrupts an eyelike shape. This suggests that eyebars provide an effective way to conceal the eyes and consequently obstruct detection and recognition of prey. This experiment also demonstrates that through concealment of the eyes, eyebars could be used to enhance the divertive effect of eyespots, which can explain the common occurrence of eyebars in many species of fish that have eyespots. Larger eyespots have been shown to intimidate some terrestrial predators, such as passerine birds, either because they resemble the eyes of the predator’s own enemy or because highly salient features may have an intimidating effect. In papers II and IV, I investigated whether the occurrence of eyespots in some aquatic prey could be explained by their intimidating effect predatory fish. In paper IV, I also investigated the reason for the intimidating effect of eyelike prey marks. In paper II, I found no clear intimidating effect of eyespots, whereas in paper IV, using a different approach, I found that sticklebacks hesitated to attack towards eyelike but not towards non-eyelike marks. Importantly, paper IV therefore presents the first rigorous evidence for the idea that eye mimicry, and not merely conspicuousness, underlies the intimidating effect. It also showed that the hesitation shown by fish towards eyelike marks is partly an innate response that is reinforced by encounters with predators. Collectively, this thesis shows that prey colour pattern and the visual appearance of the habitat influence the behaviour of fish. The results demonstrate that protective coloration provides numerous distinctive ways for aquatic prey to escape predation. Thus, visual perception and behaviour of fish are important factors shaping the appearance and behaviours of aquatic prey.

Aquatic plant community in porto primavera reservoir

Aiming to identify the populations of aquatic plants present in the Porto Primavera reservoir and evaluate the behavior of Hydrilla verticillata colonization of this water body a survey was carried out in 2007. The data was based on presence or absence, only were assessed the presence or absence of the species and the data were subjected to cluster analysis to establish differences in distribution and occurrence of populations. The community of aquatic plants showed 24 species distributed in 16 botanical families. Cyperaceae and Pontederiaceae were the most representative in terms of species richness. The submerged macrophyte Hydrilla verticillata showed the highest frequency of occurrence in the water body, showing a different behavior from the other populations of the water body. Species like Eichhornia crassipes, Eichhornia azurea, Typha dominguensis and Oxycaryum cubense also showed different behavior in relation to other populations within the community, forming large populations in lagoons and backwater areas.

Use of grass carp (Ctenopharyngodon idella) as a biological control agent for submerged aquatic macrophytes

This study aimed to evaluate feed preference and control efficacy of grass carp (Ctenopharyngodon idella) on the aquatic macrophytes Ceratophyllum demersum, Egeria densa and Egeria najas. An experiment was carried out at mesocosms conditions with 2,000 liters capacity and water residence time of 2.8 days. C. demersum, E. densa e E. najas biomasses were offered individually with sixty g and coupled in similar quantities of 30 g of each species, evaluated during 81 days, envolving 6 treatments. (1 - C. demersum, 2 - E. najas, 3 -E. densa, 4 - C. demersum + E. najas, 5 - C. demersum + E. densa and 6 - E. najas + E. densa). When offered individually, E. najas and C. demersum presented the same predation rate by grass carp, which was higher than E. densa predation rate. When plants were tested in pairs, the order of feed preference was C. demersum > E. najas > E. densa. E. najas and C. demersum percentage control ranged from 73 to 83%. No relation between biomass consumption and grass carp body weight gain was observed, probably due to differences in nutritional quality among macrophyte species according to fish necessities. Therefore, it is concluded that the use of grass carp is one excellent technique to control submersed macrophytes in Brazil.

FOOD PREFERENCE AND CONSUMPTION OF AQUATIC MACROPHYTES SUBMERGED BY SNAIL Pomacea canaliculata

ABSTRACTThe objective of this study was to evaluate the consumption potential, food preference and use of snail Pomacea canaliculata as a biocontrol agent of four submerged aquatic macrophytes (Ceratophyllumdemersum, Egeriadensa, Egerianajas and Hydrilla verticillata). Two experiments were performed. In the first experiment, the introduction of a snail took place and 10 grams of each macrophyte in plastic containers with 1 liter of water. The assessments of consumption by the snail were performed at each 48 hours, during 12 days. The second experiment was performed in 600 liters microcosms containing five snails in each experimental unit. Fifty grams of each macrophyte were offered the snails at the same time, adding the same amounts after seven, 14, 21 and 30 days. On both trials, the most consumed macrophyte by the P.canaliculata was H.verticillata (7.64 ± 1.0 g 48 h and 50 ± 0.18 g) respectively, significantly differing from the others. However, in the absence of H.verticilata, E.najas and E.densa were consumed. The preference of P.canaliculata for H.verticillata is very interesting, because this plant is exotic and problematic in Brazil, and the snail is one more tool for biological management of submerged aquatic macrophyte H.verticillata.

Interaction between the mineral content and the occurrence number of aquatic fungi in leaves submerged in a stream in the Atlantic rainforest, São Paulo, Brazil

Leaves of Ficus microcarpa L. f., Quercus robur L., and Alchornea triplinervia (Spreng.) Muell. Arg., submerged in a stream of the Atlantic rainforest in the "Reserva Biológica do Alto da Serra de Paranapiacaba", State of São Paulo, Brazil, were collected monthly, from April to November 1990, in order to determine the number of fungal occurrences (zoosporic fungi and aquatic Hyphomycetes), and the content of total N (%), total P (%), K+ (%), Ca+2 (%), Mg+2 (%), S+3 (%), Fe+3 (ppm), Cu+3 (ppm), Mn+2 (ppm), Zn+2 (ppm), Bo (ppm), Na+2 (ppm) and Al+3 (ppm). According to the tests of Kruskal-Wallis, Mann-Whitney and Wilcoxon, the means of the mineral content of the three types of leaves were significantly different, except for Mg+2 (%), Mn+2 (ppm), Zn+2 (ppm) and Na+2 (ppm). On comparing the mineral content with the number of fungal occurrence, an independence test showed a positive correlation between the presence of zoosporic fungi on the leaves of A. triplinervia and the total nitrogen, phosphorus and S+3 content, whereas the aquatic Hyphomycetes depended on the amount of Ca+2 available. Regarding leaves of F. microcarpa, the occurrence of zoosporic fungi was linked to the S+3 level, and the presence of aquatic Hyphomycetes, to the content of K+, Ca+2, S+3 and Bo. On Q. robur leaves, zoosporic fungi showed a positive correlation with the Ca+2 content, but a negative one with Fe+3 and Al+3 levels, while the occurrence of aquatic Hyphomycetes was influenced by the content of Ca+2, Mg+2, Fe+3, Al+3, Mn+2, Zn+2 and Na+2. The correlation between the occurrence number of aquatic Hyphomycetes and a high mineral content indicates that their nutritional requirements may be more complex than those of zoosporic fungi. Further studies are still required to understand the implications of this tendency on the diversity of aquatic native mycota.

Effect of nitrate and ammonium on the growth and protein concentration of Microcystis viridis Lemmermann (Cyanobacteria)

Cyanobacteria are a very important group in aquatic systems, particularly in eutrophic waters. Therefore studies about their success in the environment are essential. Many hypotheses have tried to explain the dominance of Cyanobacteria, and several emphasized the importance of various nitrogen sources for the success of the group. In this study, we measured the effect of ammonium and nitrate on the growth and protein concentration of Microcystis viridis (Cyanobacteria). This species is well-known because bloom formation in eutrophic waters. The study was carried out, in experimental batch cultures, using the WC medium with different nitrogen sources: ammonium, nitrate, ammonium + nitrate (50% ammonium + 50% nitrate) and ammonium at different concentrations (to test for possible NH4+ toxicity). Protein, ammonium and nitrate concentrations were measured at end of each experiment, whereas samples for cell counts were taken daily. Results showed that Microcystis viridis grew faster with ammonium (µ = 0.393 day-1) than with nitrate (µ = 0.263 day-1) and ammonium + nitrate (µ = 0.325 day-1). This pattern is explained by the metabolism of ammonium that presents higher uptake and assimilation rates than nitrate. Maximum cell concentration, however, was higher in the ammonium + nitrate treatment, followed by nitrate treatment. Higher protein concentration were observed in the treatment with nitrate. In the ammonium toxicity test, no difference between the control and NH4+ at 50% was found. Thus, the ammonium concentrations used in these experiments were not toxic. Our results suggest that Cyanobacteria is able to grow on both nitrogen sources even if ammonium may allow faster growth and bloom development.

Aquatic Hyphomycetes in the Parque Estadual das Fontes do Ipiranga - PEFI, São Paulo, Brazil

In the State Park Parque Estadual das Fontes do Ipiranga - PEFI, located in the city of São Paulo, samples of submerged mixed leaf litter were collected monthly from 10 sites with different levels of eutrophication, from October 2003 to April 2005. Some abiotic factors, such as pH, temperature, conductivity and dissolved oxygen were simultaneously measured in 20 cm deep water at each site. The leaf litter samples were washed in laboratory, cut into 1 cm² pieces and incubated in Petri dishes containing distilled sterile water for five to seven days at 15 °C to 20 °C. Twenty-four aquatic Hyphomycete species were identified, with predominance of Anguillospora crassa Ingold, Lunulospora curvula Ingold, Tetrachaetum elegans Ingold and Camposporium pellucidum (Grove) Hughes. First reports for South America are: Anguillospora filiformis Greathead, Dendrospora erecta Ingold and Pyramidospora casuarinae Nilsson. These species, as well as Tetracladium setigerum (Grove) Ingold, Tricladium splendens Ingold and Varicosporium elodeae Kegel, are reported for the first time in Brazil. According to the multivariate analysis, the occurrence of the aquatic Hyphomycetes was mainly influenced by the trophic level of the aquatic environments.

Floristic surveys of aquatic macrophytes in reservoirs in the Agreste zone of Pernambuco State, Brazil

We surveyed the aquatic macrophytes in reservoirs in Camocim de São Félix in the Agreste zone of Pernambuco State, Brazil. Plants were collected on a bimonthly schedule from November/2008 to May/2009 in four reservoirs, and 33 taxa distributed among 23 families were encountered. The family Cyperaceae had the greatest number of species, followed by Lemnaceae. The predominant biologic form was emergent (33.3%) reflecting the extensive shorelines around the reservoirs analyzed.

Study of the embryofeto-toxicity of Crown-of-Thorns (Euphorbia milii) latex, a natural molluscicide

The crude latex of Crown-of-Thorns (Euphorbia milii var. hislopii) is a potent plant molluscicide and a promising alternative to the synthetic molluscicides used in schistosomiasis control. The present study was undertaken to investigate the embryofeto-toxic potential of E. milii latex. The study is part of a comprehensive safety evaluation of this plant molluscicide. Lyophilized latex (0, 125, 250 and 500 mg/kg body weight) in corn oil was given by gavage to Wistar rats (N = 100) from days 6 to 15 of pregnancy and cesarean sections were performed on day 21 of pregnancy. The numbers of implantation sites, living and dead fetuses, resorptions and corpora lutea were recorded. Fetuses were weighed, examined for external malformations, and fixed for visceral examination, or cleared and stained with Alizarin red S for skeleton evaluation. A reduction of body weight minus uterine weight at term indicated that E. milii latex was maternally toxic over the dose range tested. No latex-induced embryolethality was noted at the lowest dose (125 mg/kg) but the resorption rate was markedly increased at 250 mg/kg (62.5%) and 500 mg/kg (93.4%). A higher frequency of fetuses showing signs of delayed ossification (control: 17.4%; 125 mg/kg: 27.4% and 250 mg/kg: 62.8%; P<0.05 vs control) indicated that fetal growth was retarded at doses ³ 125 mg latex/kg body weight. No increase in the proportion of fetuses with skeletal anomalies was observed at the lowest dose but the incidence of minor skeletal malformations was higher at 250 mg/kg body weight (control: 13.7%; 125 mg/kg: 14.8%; 250 mg/kg: 45.7%; P<0.05 vs control). Since a higher frequency of minor malformations was noted only at very high doses of latex which are embryolethal and maternally toxic, it is reasonable to conclude that this plant molluscicide poses no teratogenic hazard or, at least, that this possibility is of a considerably low order of magnitude

AQUAREL CONCEPT Aquatic resources for green energy realization

The AQUAREL project studied the availability and optional utilization methods for fish processing side streams and other aquatic biomaterial in the Republic of Karelia. Additionally processing aquatic biomaterial with manure and sewage sludge was studied. Based on the results, the most feasible option today is to process fish side streams to fish oil and dewatered oil-free residue and to use them for fish or animal feed production. However, it is necessary to highlight, that changes in e.g. economic environment, energy prices and demand may require re-evaluating the results and conclusions made in the project. Producing fish oil from fish processing side streams is an easy and relatively simple production process generating a valuable end product. The functionality of the process was confirmed in a pilot conducted in the project. The oil and solids are separated from the heated fish waste based on gravity. The fish oil separating on top of the separator unit is removed. Fish oil can as such be utilized for heating purposes, fish meal or animal feed production, but it can also be further processed to biodiesel. However, due to currently moderate energy prices in Russia, biodiesel production is not economically profitable. Even if the fish oil production process is not complicated, the operative management of small-scale fish oil production unit requires dedicated resources and separate facilities especially to meet hygiene requirements. Managing the side streams is not a core business for fish farmers. Efficient and economically profitable fish oil production requires a centralized production unit with bigger processing capacity. One fish processing unit needs to be designed to manage side streams collected from several fish farms. The optimum location for the processing unit is in the middle of the fish farms. Based on the transportation cost analysis in the Republic of Karelia, it is not economically efficient to transport bio-wastes for more than 100 km since the transportation costs start increasing substantially. Another issue to be considered is that collection of side streams, including the dead fish, from the fish farms should be organized on a daily basis in order to eliminate the need for storing the side streams at the farms. Based on AQUAREL project studies there are different public funding sources available for supporting and enabling profitable and environmentally sustainable utilization, research or development of fish processing side streams and other aquatic biomaterial. Different funding programmes can be utilized by companies, research organizations, authorities and non-governmental organizations.

Genetic responses against nitric oxide toxicity

The threat of free radical damage is opposed by coordinated responses that modulate expression of sets of gene products. In mammalian cells, 12 proteins are induced by exposure to nitric oxide (NO) levels that are sub-toxic but exceed the level needed to activate guanylate cyclase. Heme oxygenase 1 (HO-1) synthesis increases substantially, due to a 30- to 70-fold increase in the level of HO-1 mRNA. HO-1 induction is cGMP-independent and occurs mainly through increased mRNA stability, which therefore indicates a new NO-signaling pathway. HO-1 induction contributes to dramatically increased NO resistance and, together with the other inducible functions, constitutes an adaptive resistance pathway that also defends against oxidants such as H2O2. In E. coli, an oxidative stress response, the soxRS regulon, is activated by direct exposure of E. coli to NO, or by NO generated in murine macrophages after phagocytosis of the bacteria. This response is governed by the SoxR protein, a homodimeric transcription factor (17-kDa subunits) containing [2Fe-2S] clusters essential for its activity. SoxR responds to superoxide stress through one-electron oxidation of the iron-sulfur centers, but such oxidation is not observed in reactions of NO with SoxR. Instead, NO nitrosylates the iron-sulfur centers of SoxR both in vitro and in intact cells, which yields a form of the protein with maximal transcriptional activity. Although nitrosylated SoxR is very stable in purified form, the spectroscopic signals for the nitrosylated iron-sulfur centers disappear rapidly in vivo, indicating an active process to reverse or eliminate them.

Toxicity to sea urchin egg development of the quinone fraction obtained from Auxemma oncocalyx

Auxemma oncocalyx Taub. belongs to the Boraginaceae family and is native to the Brazilian northeast where it is known as "pau-branco". We investigated the ability of the water soluble fraction isolated from the heartwood of A. oncocalyx to inhibit sea urchin egg development. This fraction contains about 80% oncocalyxone A (quinone fraction), a compound known to possess strong cytotoxic and antitumor activities. In fact, the quinone fraction inhibited cleavage in a dose-dependent manner [IC50 of 18.4 (12.4-27.2) µg/ml, N = 6], and destroyed the embryos in the blastula stage [IC50 of 16.2 (13.7-19.2) µg/ml, N = 6]. We suggest that this activity is due to the presence of oncocalyxone A. In fact, these quinones present in A. oncocalyx extract have strong toxicity related to their antimitotic activity.

Toxicity of a cyanobacterial extract containing microcystins to mouse lungs

Toxic cyanobacteria in drinking water supplies can cause serious public health problems. In the present study we analyzed the time course of changes in lung histology in young and adult male Swiss mice injected intraperitoneally (ip) with a cyanobacterial extract containing the hepatotoxic microcystins. Microcystins are cyclical heptapeptides quantified by ELISA method. Ninety mice were divided into two groups. Group C received an injection of saline (300 µl, ip) and group Ci received a sublethal dose of microcystins (48.2 µg/kg, ip). Mice of the Ci group were further divided into young (4 weeks old) and adult (12 weeks old) animals. At 2 and 8 h and at 1, 2, 3, and 4 days after the injection of the toxic cyanobacterial extract, the mice were anesthetized and the trachea was occluded at end-expiration. The lungs were removed en bloc, fixed, sectioned, and stained with hematoxylin-eosin. The percentage of the area of alveolar collapse and the number of polymorphonuclear (PMN) and mononuclear cell infiltrations were determined by point counting. Alveolar collapse increased from C to all Ci groups (123 to 262%) independently of time, reaching a maximum value earlier in young than in adult animals. The amount of PMN cells increased with time of the lesion (52 to 161%). The inflammatory response also reached the highest level earlier in young than in adult mice. After 2 days, PMN levels remained unchanged in adult mice, while in young mice the maximum number was observed at day 1 and was similar at days 2, 3, and 4. We conclude that the toxins and/or other cyanobacterial compounds probably exert these effects by reaching the lung through the blood stream after ip injection.