The use of clove oil to induce anaesthesia,and its effects on blood chemistry,in Lates niloticus from Lake Victoria,Uganda

The efficacy of clove oil as an anaesthetic and its effects on blood parameters in Nile perch Lates niloticus were evaluated in 2010. Clove oil concentrations of 49.3, 73.9 and 98.5?mg l^?1 induced anaesthesia in <3?min, while the average recovery time from anaesthesia was 11?min 22 s. The optimal oil clove oil concentration was 49.3?mg l^?1, inducing anaesthesia in 4?min 33 s, with recovery in 3?min 31 s. No stress response was elicited. Clove oil at a concentration of 24.6?mg l^?1 was an effective sedative, whereas a concentration of 49.3?mg l^?1 was sufficient for measuring fish and stripping gametes. A concentration of 73.9?mg l^?1 induced anaesthesia within 4?min and fish recovered in 10?min. Therefore, clove oil was an effective anaesthetic and sedative for the handling of Nile perch within a mass range of 0.4–12?kg fish^?1.     

Testing clove oil as an anaesthetic for long-distance transport of live fish: the case of the Lake Victoria cichlid Haplochromis obliquidens

Clove oil can be used as an anaesthetic in the handling of marine and freshwater fish. Few studies report on its use for periods up to 48 h, for example, under long‐distance transport conditions. This study tested the effect of different clove oil concentrations for 1–48 h on recovery and survival of the cichlid Haplochromis obliquidens, an ornamental fish species endemic to Lake Victoria. Haplochromis obliquidens were anaesthetized for 1 h using 5–25 μl L^?1 clove oil. There was no correlation between clove oil concentration and post‐anaesthesia recovery time (P = 0.15). On average, fish recovered within 9.5 ± 2 min, and no fish died within 24 h after recovery. Results from exposure of fish to 18–20 μl L^?1 clove oil for up to 48 h suggested a narrow margin of safety as this concentration range induced mortality. At 18 μl L^?1 recovery times ranged from 3 to 43 min between 24 and 36 h exposure, while fish exposed longer than 36 h recovered within 1–10 min, or within 1–2 min after 44–48 h. At the end of a 48‐h transport experiment total ammonia levels were higher in transport water containing anaesthetized fish than for non‐anaesthetized fish (1.65 ± 0.19 and 0.54 ± 0.08 mg L^?1 NH + NH₃, respectively). The combined use of clove oil and the selective ammonium ion exchanger zeolite was considered feasible as ammonia levels could be reduced by up to 82% compared to control bags without zeolite.     

Effects of 2‐phenoxyethanol anaesthesia on juvenile meagre (Argyrosomus regius)

This study was performed to evaluate the effective concentration of the anaesthetic 2‐phenoxyethanol (2‐PE) on juvenile (1.3 ± 0.03 g) meagre (Argyrosomus regius, Asso, 1801) and establish the LC₅₀ (through a series of exposure concentrations) and LT₅₀ of 2‐PE at 20 ± 0.5°C, salinity 38 g × L^?1, pH 8.2–8.4 and dissolved oxygen >7 mg × L^?1. The induction time decreased and the recovery time increased with increasing concentrations. Conflicting results were found only in recovery time and there were no significant differences among the recovery times from all concentrations. The most suitable concentration of 2‐PE was 0.3 ml × L^?1 for about or over 15 min exposure time. The LC₅₀ and LT₅₀ for the 3–60 min exposure periods were estimated for juvenile meagre. The toxic effect of 2‐PE on survival rates of A. regius juveniles increased depending on the exposure period. In addition, 2‐phenoxyethanol LT₅₀ (median survival time) values, slope function (S) and lower and upper 95% confidence limits were estimated.     

Menthol as an alternative anaesthetic and sedative for rainbow trout,Oncorhynchus mykiss

Menthol is known for its analgesic properties, but relatively little information is available on its potential as an anaesthetic on fish. The purpose of this study was to assess anaesthetic and sedative effectiveness of menthol and its safety in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout 180 ± 28 g (mean ± SD) within a range of 152–208 g fish^?1 were individually exposed to menthol concentrations between 10 and 150 mg l^?1 and observed for behavioural responses, induction time to anaesthesia and recovery time. Menthol concentrations of 40–150 mg l^?1 induced anaesthesia with varying exposure times. There was an exponential relationship (p < 0.001) between induction time and menthol concentration. Menthol concentrations of 80–150 mg l^?1 induced anaesthesia within three minutes of exposure and fish recovered within three minutes. Induction and recovery data showed that 80 mg l^?1 was most suitable for anaesthesia in juveniles of this species. Concentrations of 10–20 mg l^?1 had sedative effects. Menthol stock solutions prepared using ethanol and acetone and storage time of stock solutions at room temperature for up to 48 h showed no significant differences in anaesthetic efficiency. When exposure time to menthol was kept constant at three minutes, 22% of fish had temporary cessation of gill ventilation. These fish had longer recovery times than those that did not show that response. Menthol was an effective anaesthetic and could be tested as a sedative for trout.     

Effects of clove oil,essential oil of Lippia alba and 2‐phe anaesthesia on juvenile meagre,Argyrosomus regius (Asso, 1801)

The objectives of this experiment were to (i) determine the efficacy of essential oils of clove (CO) and Lippia alba (EOLA) to induce deep anaesthesia in juvenile specimens (49.0 ± 6.2 g body mass, 16.6 ± 0.8 cm; n = 8 per treatment) of meagre (Argyrosomus regius); and (ii) study the feasibility of these substances, together with 2‐phenoxyethanol (2‐PHE), as potential sedatives [low concentration: (i) EOLA: 12 mg L^?1; (ii) CO: 1 mg L^?1; and (iii) 2‐PHE: 33 mg·L ^?1; n = 8 per treatment] for live fish transport of this species. All test were performed at a constant temperature (18°C). Thus, the main primary stress indicator (plasma cortisol) and secondary factors (plasma metabolites) were evaluated. In addition, growth hormone (GH) mRNA expression was also evaluated in the pituitary gland. The results indicated that EOLA is considered to be effective for deep anaesthesia when the concentration is close to 160 mg L^?1, while CO produces the same effect when lower concentrations are added (40–50 mg L^?1). Regarding sedative concentrations, a significant ~3‐fold increase in plasma cortisol levels was detected in the EOLA group when compared to control specimens. In addition, glucose levels were not reduced and significantly increased (~1.6‐fold) for 2‐PHE in relation to the control fish. None of the anaesthetics promoted a significant difference for GH expression with respect to the control group, but a significant ~2‐fold increase for 2‐PHE treatment with respect to the EOLA exposition was found in this gene expression. Results show that none of the anaesthetics analysed, at least in the ranges of concentrations used in this study (EOLA 12 mg L^?1, CO 1 mg L^?1, 2‐PHE 33 mg L^?1), are recommended for live fish transport, as shown by the absence of inhibition on the stress parameters assessed.     

Simultaneous Removal of Mn(II) and Nitrate by the Manganese-Oxidizing Bacterium Acinetobacter sp. SZ28 in Anaerobic Conditions

A novel bacterium, strain SZ28, identified as Acinetobacter sp., showed anaerobic denitrification ability using Mn(II) as the electron donor. Nitrate-nitrogen concentration decreased from nearly 16.52–mg L^?1 to 4.4–mg L^?1, without accumulation of nitrite as an intermediate, with a maximum of 0.063–mg NO₃^?-N L^?1 h^?1, reaching a peak of 0.085–mg NO₃^?-N L^?1 h^?1 in sodium acetate. The nitrate removal rate reached 0.067–mg NO₃^?-N L^?1 h^?1, 0.059–mg NO₃^?-N L^?1 h^?1, and 0.078 mg NO₃^?-N L^?1 h^?1 using Mn(II), S(II), and Fe(II) as electron donors, respectively. The optimum pH was 6.0, with a removal rate of 0.063–mg NO₃^?-N L^?1 h^?1     

Anaesthetic effects of clove oil on seven species of tropical reef teleosts

The anaesthetic potential of the clove oil was tested on the following species of tropical reef fishes: Abudefduf saxatilis , Stegastes variabilis , Pareques acuminatus , Acanthurus chirurgus , Sparisoma axillare , Lutjanus apodus and Bathygobius soporator . Induction and recovery times from anaesthesia were compared using various concentrations (20, 30, 40, 50 and 60 mg l⁻¹). Induction and recovery times were not affected by variations in fish total length. When exposed to any of the five tested concentrations of clove oil, specimens achieved a deep state of anaesthesia, with induction and recovery times of <180 and <300 s, respectively. Nevertheless, to maximize safety and reduce fish mortality and stress, the lowest concentration (20 mg l⁻¹) is recommended during field sampling.     

Temperature effects on anaesthesia with clove oil in six temperate‐zone fishes

The potential use of clove oil (eugenol) as an anaesthetic for Atlantic salmon Salmo salar , brown trout Salmo trutta , rainbow trout Oncorhynchus mykiss , whitefish Coregonus lavaretus , perch Perca fluviatilis and roach Rutilus rutilus was examined at 5, 10, 15 and 20° C using three anaesthetic concentrations (varying from 20 to 200 mg l⁻¹) at each temperature. Substantial species differences in sensitivity to clove oil were observed, even amongst congeners, and there may be some disadvantages (slow recovery and possibly mortality) with using clove oil for 0+ year whitefish and at low temperatures for perch and roach.     

One-stage partial nitritation/anammox at 15 °C on pretreated sewage: feasibility demonstration at lab-scale

Energy-positive sewage treatment can be achieved by implementation of oxygen-limited autotrophic nitrification/denitrification (OLAND) in the main water line, as the latter does not require organic carbon and therefore allows maximum energy recovery through anaerobic digestion of organics. To test the feasibility of mainstream OLAND, the effect of a gradual temperature decrease from 29 to 15 °C and a chemical oxygen demand (COD)/N increase from 0 to 2 was tested in an OLAND rotating biological contactor operating at 55–60 mg NH₄ ⁺–N?L^?1 and a hydraulic retention time of 1 h. Moreover, the effect of the operational conditions and feeding strategies on the reactor cycle balances, including NO and N₂O emissions were studied in detail. This study showed for the first time that total nitrogen removal rates of 0.5 g N?L^?1?day^?1 can be maintained when decreasing the temperature from 29 to 15 °C and when low nitrogen concentration and moderate COD levels are treated. Nitrite accumulation together with elevated NO and N₂O emissions (5 % of N load) were needed to favor anammox compared with nitratation at low free ammonia (<0.25 mg N?L^?1), low free nitrous acid (<0.9 μg N?L^?1), and higher DO levels (3–4 mg O₂?L^?1). Although the total nitrogen removal rates showed potential, the accumulation of nitrite and nitrate resulted in lower nitrogen removal efficiencies (around 40 %), which should be improved in the future. Moreover, a balance should be found in the future between the increased NO and N₂O emissions and a decreased energy consumption to justify OLAND mainstream treatment.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of GA<Subscript>3</Subscript> on morphogenesis of CIP potato explants and acclimatization of plantlets in field

Improvement of potato has been accomplished using conventional and non-conventional approaches coupled with numerous tissue culture procedures. The aim of the present study was to assess the efficacy of gibberellic acid (GA₃) on the morphogenesis of International Potato Center (CIP) potato explants and acclimatization of plantlets in the field. Nodal segments as an explant source (1–1.5 cm) were isolated from 31 CIP potato plantlets and were inoculated into Murashige and Skoog (MS) medium supplemented with 0.0 (control), 0.1, 0.5, or 1.0 mg L^?1of GA₃. The variation in growth parameters of the cultivars was then observed. The highest shoot induction occurred in MS medium containing 1.0 mg L^?1 GA₃ with an increase in the inter-nodal distance between nodes as compared to other treatments. Higher concentration (1.0 mg L^?1) of GA₃ significantly increased plant height and root length in the treated germplasm however; this concentration was inhibitory to the number of nodes and roots per plant. The number of leaves was significantly increased in plants receiving GA₃ treatment at lower concentration (0.1 mg L^?1). The 31 CIP genotypes were transplanted to the field and checked for yield quality traits. It was concluded from the results that GA₃ had significant effects on morphogenesis and was effective in the acclimatization of CIP potato plantlets in field.     

Comparative efficacy of clove oil and other chemicals in anaesthetization of Pomacentrus amboinensis, a coral reef fish

The efficacy of quinaldine, benzocaine, MS-222, 2-phenoxyethanol and clove oil was compared for anaesthetizing settlement stage Pomacentrus amboinensis , a frequently studied coral reef fish. Induction to anaesthesia, behaviour during anaesthesia, recovery times and survival rates of fish treated with the different chemicals were compared. Clove oil was only marginally less effective than quinaldine and was more effective than other chemicals tested, except at high concentrations. In addition, fish exposed to clove oil exhibited a much calmer induction to anaesthesia than fish exposed to quinaldine. Therefore, clove oil may be an effective alternative to quinaldine as a fish anaesthetic. Recovery time after anaesthesia with clove oil was two to three times longer than recovery from other chemicals, a desirable charcteristic for use in field studies. Survival rates were excellent for all chemicals.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20–60°C) and protein concentration (5–100 µmol L^?1). The time courses of loss of enzyme activity in 100 mmol L^?1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k_a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k_b) and binding (k_?b) as well as the irreversible aggregation of the apo-enzyme (k_agg) were less sensitive to changes in temperature, their activation energy (E_a) being about 52 kJ mol^?1 in comparison with an E_a value of 185 kJ mol^?1 for k_a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5′-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^?1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

The Effects of Selenium on Physiological Traits, Grain Selenium Content and Yield of Winter Wheat at Different Development Stages

The paper evaluated the effects of Se application time and rate on physiological traits, grain Se content, and yield of winter wheat by field experiment. Se application significantly increased grain Se content and yield, and the increased amount treated with 20 and 30 mg Se?L^?1 was the highest. At blooming–filling stage, Se application significantly increased grain Se content, but did not affect yield. Chlorophyll content was increased by Se application, and the increased amount at heading–blooming stage was higher than that in wheat leaves at the other stages. At four development stages, Se treatments (except for 10 mg Se?L^?1 at jointing–heading stage) significantly decreased the rate of superoxide (O₂ ^?) radical production. At heading–blooming (except for 50 mg Se?L^?1) and blooming–filling stages, hydrogen peroxide (H₂O₂) content was significantly decreased by Se treatments. The rate of O₂ ^? production and H₂O₂ content at 20 and 30 mg Se?L^?1 was the lowest. Se treatments (except for 10 mg Se?L^?1 at regreening–jointing and blooming–filling stages) also induced an evident decrease in malondialdehyde content. Proline content induced by Se treatments at jointing–heading and heading–blooming stages was higher than that in wheat leaves at regreening–jointing and blooming–filling stages. At four development stages, Se treatments all significantly increased glutathione peroxidase activity, and the treatments with 20 and 30 mg Se?L^?1 also evidently increased reduced glutathione content. These results suggested that Se application at different development stages increased antioxidant capacity of wheat, reduced oxidant stress to some extent, and the effects of Se treatments was the best if Se concentration ranged between 20 and 30 mg Se?L^?1. In addition, Se application time was more beneficial for Se accumulation and yield in wheat grain at heading–blooming stage.     

Identification of somaclonal variants in proliferating shoot cultures of Senecio cruentus cv. Tokyo Daruma

A protocol for in vitro propagation of cineraria (Senecio cruentus) was developed. The highest frequency of shoot proliferation was obtained from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with 2.0?mg L^?1 6-benzyladenine (BA) and 0.5?mg L^?1 ??-naphthalene acetic acid (NAA), with a mean number of 14 shoots per explant. A high concentration of BA (4.0?mg L^?1) and repeated subcultures resulted in hyperhydric shoots. Decreasing the BA concentration to 1.0?mg L^?1 in the culture medium eliminated hyperhydricity. The concentration of ammonium nitrate (NH₄NO₃) and temperature had marked effects on somaclonal variation. Variation was observed when the cultures were maintained at 15?°C but not at 25?°C. Variants with blue-colored leaves and stems were identified; whereas, normal plants maintained their green-colored leaves and stems. The highest frequency of variation (67.5?%), with a mean number of 3.0 variant shoots per explants, was obtained on shoot proliferation medium (MS?+?2.0?mg L^?1 BA and 0.5?mg L^?1 NAA) devoid of NH₄NO₃. The best rooting (100?%), with the highest number of roots per shoot (10.8) and the greatest root length (6.8?cm) was obtained on medium supplemented with 0.1?mg L^?1 NAA. In vitro-grown plantlets were successfully acclimatized in a greenhouse, and transferred to the field.     

Effects of different concentrations of 2‐phenoxyethanol on primary and secondary stress responses in Persian sturgeon,Acipenser persicus

The anaethetic effects of 2‐phenoxyethanol (2‐PE) on possible primary (cortisol level) and secondary (hematological indices and glucose level) stress responses were studied in Persian sturgeon (Acipenser persicus). Fish were first exposed to 0.1, 0.3, 0.5, 0.7, 0.9 and 1.1 ml L^?1 2‐PE, and the time to induction (deep anaethesia) and recovery were measured. At a concentration of 0.1 ml L^?1, 2‐PE failed to induce deep anaethesia in fish, whereas at concentrations of 0.7, 0.9 and 1.1 ml L^?1 all fish were anaethetized within 3 min of exposure. For assessing possible stress effects caused by effective concentrations of 2‐PE, the hematological indices, serum cortisol and glucose were determined in the deeply anaethetized fish as stress indicators. The 2‐PE exposure resulted in significant increases in red blood cell (RBC) values at 0.3 and 0.5 ml L^?1; parallel increases in hemoglobin values were also observed at these concentrations (P < 0.01). Moreover, a lower concentration of 2‐PE (0.3 ml L^?1) caused a significant increase in hematocrit values (P < 0.05). Among the hematological indices, mean corpuscular volume (MCV) values decreased at 0.5 ml L^?1 when compared with the control and other groups (P < 0.05). Serum cortisol level was elevated at 0.3, 0.5 and 0.7 concentrations of 2‐PE (P < 0.01). The glucose level followed a trend similar to that observed for cortisol. The outcome of these experiments shows that 2‐PE at a concentration of 0.9 ml L^?1 is a suitable anaesthetic for Persian sturgeon. This study demonstrates that rapid induction of deep anaethesia with a relatively high concentration of 2‐PE (0.9 and 1.1 ml L^?1) was associated with the lowest effects on signs of physiological stress in Persian sturgeon.     

Effect of temperature on ethanol tolerance of thermotolerant Isshatchenkia orientalis IPE100

Tolerance to high temperature and ethanol is a major factor in high‐temperature bio‐ethanol fermentation. The inhibitory effect of exogenously added ethanol (0–100 g L^?1) on the growth of the newly isolated thermotolerant Issatchenkia orientalis IPE100 was evaluated at a range of temperatures (30–45°C). A generalized Monod equation with product inhibition was used to quantify ethanol tolerance, and it correlated well with the experimental data on microbial growth inhibition of ethanol at the temperatures of 30–45°C. The maximum inhibitory concentration of ethanol for growth (P_m) and toxic power (n) at the optimal growth temperature of 42°C were estimated to be 96.7 g L^?1 and 1.23, respectively. The recently isolated thermotolerant I. orientalis IPE100 shows therefore a strong potential for the development of future high‐temperature bio‐ethanol fermentation technologies. This study provides useful insights into our understanding of the temperature‐dependent inhibitory effects of ethanol on yeast growth.     

Clove oil as an anaesthetic for adult sockeye salmon: field trials

Wild migrating sockeye salmon Oncorhynchus nerka exposed to 20, 50 and 80 mg 1⁻¹ of clove oil could be handled within 3 min, recovered within 10 min, and survived 15 min exposure trials. Fish tested at 110 mg 1⁻¹ did not recover from 15 min exposure trials. Response curves developed for induction and recovery time considered the following predictors: clove oil concentration, sex, fish length and depth. A significant positive dependence was observed between induction time and fish length for 20, 50 and 80 mg 1⁻¹ test concentrations; no dependence was observed between induction time and length at 110 and 140 mg 1⁻¹. Recovery time differed as a function of clove oil concentration, but not fish size. A concentration of 50 mg 1⁻¹ is recommended for anaesthetizing sockeye salmon ranging in length from 400 to 550 mm at water temperatures averaging 9–10° C.     

The use of clove oil as an anaesthetic and method for sampling intertidal rockpool fishes

A suitable concentration of clove oil for anaesthetizing eight common Australian intertidal fishes was 40 mg l⁻¹. Induction and recovery times at this concentration varied markedly interspecifically, although were <3 and 5 min, respectively for all but Enneapterygius rufopileus . The number of species in clove oil samples was remarkably similar to bilge pumping samples in both small and large pools, although the number of individuals was significantly lower than bilge pumping in large pools. Number of species and individuals recorded by visual census differed from clove oil and bilge pumping methods in both large and small pools. Failure to detect all fish in pools by clove oil sampling and visual census was due to the secretive behaviour of many species and the tendency for such species as Bathygobius cocosensis not to emerge from under rocks during induction to anaesthesia. Complete draining of pools by bilge pumping, although time consuming, was a more accurate method of sampling rockpool fish assemblages than clove oil. However, clove oil is an effective stand-alone sampling method because the number of fish and individuals are remarkably similar to samples obtained by bilge pumping. Clove oil is a favourable anaesthetic for temperate Australian intertidal fishes as mortality is extremely low, it is inexpensive and user-safe, and induction and recovery times are short, although still sufficient to record biological information and identifications of fish whilst under anaesthesia.     

Effects of stocking density on survival,growth and size variation of juvenile Brachymystax lenok (Pallas, 1773)

In this experiment the effects of stocking density on growth and survival of juvenile Brachymystax lenok were studied. Experimental fishes were reared at different stocking densities (5, 25, 50 and 75 fish L^?1) for 40 days at ambient temperatures (11.2–16.4°C). During the experiment, dissolved oxygen (DO) was above 5.4 mg L^?1 and other major environmental factors were controlled at the same levels in each aquarium (DO 5.4–7.2 mg L^?1, pH 6.6–7.4, and flow‐through rates 0.3–0.4 L s^?1). Mortality data in each group was recorded daily and random samples of 30 juveniles were measured and weighed every 10 days. All statistical analyses were performed by anova . The survival rate was found to be significantly affected by stocking density, but not the condition factor (CF), specific growth rate (SGR) or coefficients of variation (CV).The results demonstrate that lenok juveniles can be reared at higher stocking densities, which can increase survival under intensive conditions.     

Response of juvenile tench Tinca tinca (L.) to the anaesthetic 2-phenoxyethanol

The response of tench Tinca tinca aged 40–171 days post‐hatch (22–49 mm TL) to the anaesthetic 2‐phenoxyethanol was studied at 25°C. The lowest effective concentration of 2‐phenoxyethanol increased with age, while the highest safe concentration decreased. The fish aged 40 days post‐hatch required a significantly (P ≤ 0.05) shorter time to become anaesthetized than did older fish. The recovery time after 15 min of exposure to 2‐phenoxyethanol at 0.45 g dm^?3 was significantly shorter in the 40‐day‐old fish than in older fish. In juveniles of the same age, induction time or recovery time did not depend on their size or condition (Fulton's coefficient). At 25°C, 2‐phenoxyethanol at 0.5 g dm^?3 may be used to efficiently and safely anaesthetize T. tinca juveniles.