Studies on the ability of two isolates of Bacillus thuringiensis,an isolate of Clonostachys rosea f. rosea and a diatomaceous earth product to control gastrointestinal nematodes of sheep

Abstract

Nematode parasites have developed resistance to anthelmintics. Biological control of gastrointestinal nematodes (GIN) in sheep is a promising non-chemical control method. Two experiments were done using Merino sheep. In each experiment, gender, initial egg count per gram (EPG) and initial body weight (BW) aided in placing animals into four groups, each of which was randomly fed with one of four treatments. Experiment 1 evaluated anthelmintic effects of Bacillus thuringiensis Berliner (Bt), Clonostachys rosea f. rosea Schroers (C. rosea) and diatomaceous earth (DE) in sheep. Bacillus thuringiensis and C. rosea were fed to sheep at a rate of 1 g kg^?1 BW, and DE was fed at 2% of sheep diet. Relative to the control, treatments had no effect (P>0.05) on EPG, but reduced (P<0.001) larvae per gram (LPG) in faecal culture. Efficacy varied with time (P<0.001). On Day 7, Bt, C. rosea and DE had efficacies of 76, 87 and 61%, respectively. In experiment 2, efficacy of feeding 1 g kg^?1 BW of C. rosea chlamydospores to sheep every day, every second day and every third day was tested. Daily feeding of fungal chlamydospores had no effect on EPG (P>0.05), but reduced (P<0.001) LPG (12±1.67) more than every second day (39±0.77) or third day (58±1.77). On Day 12, feeding daily, every second day and every third day had efficacies of 90, 63 and 49%, respectively. These studies suggest that each of Bt, C. rosea isolates and DE products has a potential to affect nematode larvae, and daily use of C. rosea had the highest effect as a biological control of nematodes in this study.     

A new control strategy for nematodes of sheep using chlamydospores of a fungus,Clonostachys rosea f. rosea,and an ethanolic extract of a plant,Ananas comosus

An experiment was conducted at Ukulinga Research Farm using 24 Merino sheep to test a combination of plant Ananas comosus (Ac) and fungus Clonostachys rosea (Cr) in controlling nematode parasites. Four treatments were assigned: 100 mg kg^?1 body weight (BW) ethanol extract of Ac, 1 g of Cr product kg^?1 BW, the combination of both treatments (AcCr), and a Control. Sheep within a treatment were paired and penned in individual paddocks, and rectal faecal samples were taken for counting of eggs per gram (EPG) of faeces and third-stage (L3) larvae. Grass samples were taken every 21 days for counting of L3 larvae. A. comosus and AcCr reduced (P < 0.001) EPG. A. comosus, C.rosea and AcCr reduced (P < 0.001) L3 counts. The efficacy of treatments increased with time (P < 0.001). C. rosea and AcCr reduced (P < 0.001) L3 on grass. In conclusion, AcCr is better at controlling nematodes than either Ac or Cr individually.     

Effects of temperature on survival, development, growth and feeding of larvae of Yellowtail clownfish Amphiprion clarkii (Pisces: Perciformes)

To understand the physiological and ecological responses of marine fishes to the change of water temperature, newly-hatched larvae of Yellowtail clownfish Amphiprion clarkii were reared in captivity at water temperatures of 23, 26 and 29 °C till they completed the metamorphosis to juvenile phase, and larval survival, development, growth and feeding were evaluated during the experimental period. The results showed that water temperature influenced the physiological performance of larvae of A. clarkii significantly. The survival and growth rates of larvae of A. clarkii increased significantly with the increase of water temperature from 23 to 29 °C (P < 0.05). Water temperature also influenced larval development of A. clarkii significantly and larvae reared at 23 °C took longer time for post-larval development and metamorphosis compared to 26 and 29 °C (P < 0.05). Total length and body weight for post-larval development and metamorphosis decreased with the increase of water temperature from 23 to 29 °C (P < 0.05). Q₁₀ in developmental rate was higher than in daily growth rate at the same rearing temperature, indicating that at water temperature had greater influence on larval development than on growth. Water temperature also influenced larval feeding of A. clarkii significantly with feed ration (FR) and feed conversion efficiency (FCE) increased with the increase of water temperature from 23 to 29 °C (P < 0.05). There was a positive correlation between FR and specific growth rate (SGR) (P < 0.05) but not between FCE and SGR (P > 0.05), indicating that FR influenced growth rate significantly in larvae of A. clarkii. This study demonstrated that the physiological responses of larvae of A. clarkii to the change of water temperature and confirmed that water temperature influenced larval survival, development, growth and feeding significantly. This study suggests that the decline of larval survival and growth rates, extension of pelagic larval duration and reduction of larval feeding at lower temperature have ecological impacts on larval dispersal and metamorphosis, juvenile settlement and population replenishment in A. clarkii in the wild.     

Resistance to gastrointestinal parasite infection in Djallonké sheep

Gastrointestinal parasitism places serious constraints on small ruminant production. The situation has been exacerbated by development of drug resistance in many parasite populations, leading to interest in identification of animals with genetically mediated resistance or tolerance to nematode infections. This study assessed the response to natural infection with gastrointestinal nematodes (GIN) in Djallonké sheep during the rainy season in the Sudan-Guinea Savannah region of Burkina Faso. Haemonchus contortus is the most prevalent GIN at this site and time. Djallonké lambs (n=434) were sampled from 40 households and evaluated at a common location in southern Burkina Faso. Lambs were dewormed with levamisole at 2 to 6 months of age and returned to infected pastures. Fecal egg counts (FEC), packed cell volumes (PCV), and FAffa Malan CHArt (FAMACHA©) scores were determined 28 and 35 days after deworming. Lamb mortality was monitored throughout the experiment. Least-squares means for BW increased from 13.8±0.2 kg at 28 days to 14.0±0.2 kg at 35 days (P<0.01). Simple means and medians for FEC were 615 and 100, respectively, at 28 days and 850 and 175, respectively, at 35 days. The FEC exhibited strong right skewness. Following logarithmic transformation and back-transformation of resulting least-squares means to the original scale, FEC were higher (P<0.01) for males (208±27) than females (122±10). Least-squares means for PCV decreased (P<0.001) from 28 (36.3±0.5%) to 35 days (33.7±0.5%), and were higher (P<0.01) for females (36.0±0.4%) than males (33.9±0.7%). Correlations (r) between repeated measurements of BW, FEC, PCV and FAMACHA scores at 28 and 35 days were all positive (P<0.001). The correlation between FAMACHA scores and PCV was negative at 28 (r=−0.14) and 35 days (r=−0.18) (P<0.001). This study revealed that BW was an easily measured predictor of the ability of the lamb to resist infection with GIN and maintain PCV, and confirmed that FAMACHA scores are useful indicators of differences in FEC. Approximately 40% of female and 30% of male lambs did not show detectable levels of infection (i.e. FEC=0) under field conditions. The great variability that was observed in FEC and PCV suggests potential to use Djallonké sheep in breeding programs to enhance resistance to GIN.     

Application of a formulation of the nematophagous fungus <Emphasis Type="Italic">Duddingtonia</Emphasis><Emphasis Type="Italic">flagrans</Emphasis> in the control of cattle gastrointestinal nematodiosis

The viability of a formulation of Duddingtonia flagrans was assessed in the control of parasite gastrointestinal nematodes of cattle. Two groups (A and B) of eight crossbred Holstein × Zebu cattle, approximately one year old, were placed in Brachiaria decumbens pasture. Each animal in group B (treated) received orally 20 g sodium alginate pellets containing mycelial mass of the D. flagrans fungus, while the animals in the group A (control) received pellets without fungus for seven months, starting in March 2005. The egg per gram of feces counting the gastrointestinal nematodes showed a difference (P < 0.05) in the treated group in June, July and August, with reductions of 58% (June), 47% (July) and 51% (August) compared to the control group. The infective larvae recovered in the pastures collected up to 20 cm from distance of the fecal dung in group B differed (P < 0.01) from the larvae recovered in group A. At the end of the experimental period, the animals in group B presented a greater weight gain (P < 0.01) compared to the untreated group (A). The treatment of cattle with pellets containing the D. flagrans nematophagous fungus, at the dose and duration used was effective in controlling the infective larvae of gastrointestinal nematodes of cattle.     

Effect of certain herbicide treatments on pasture composition and inoculum of the take-all fungus

The improvement of pastures by the use of a range of herbicides to eliminate grasses, and their effect on populations of the take-all fungus (Gaeumannomyces graminis vartritici=Ggt) were studied in the field (at Esperance Downs, on the south-coast of Western Australia) from 1982 to 1985. Field trials were conducted to evaluate three herbicide treatments (2,4-D amine+propyzamide; 2,4-D amine+paraquat; paraquat/ diquat) and an unsprayed control. A pot trial involving these treatments with two levels of nitrogen was undertaken to confirm treatment effects observed in the field trial. All herbicide treatments resulted in reduced grass composition of pastures, in both the year of spraying and in the second year of pasture, but reduced dry matter production in the year of spraying. In the year of spraying, however, inoculum ofGgt was reduced (P<0.1) only following the 2,4-D amine+propyzamide treatment and was greater (P<0.1) after 2,4-D amine+paraquat treatment than the unsprayed treatment. Despite reduced grass levels in the herbicide-treated plots in the second year of pasture,Ggt inoculum did not differ between treatments, nor did it after a wheat crop which followed a second year pasture. There was high correlation (P<0.001) between disease levels and dry weights of grasses in the pot trial. There was significantly less (P<0.001) grass in pots treated with herbicides compared to the unsprayed control but no difference (P>0.05) was evident between treatments. Inoculum levels were lower (P<0.05) in the treated pots than the unsprayed control with no evidence of differences among treatments (P>0.05). Nitrogen level had no effect on disease (P>0.05). All herbicide treatments tested reduced grass level and total dry matter, both in the field and in pots. Whereas in the pot trial reduced grass levels resulted in reducedGgt inoculum, in the field such a reduction occurred only with the 2,4-D amine+propyzamide treatment and only in the year of spraying. Herbicide treatments had no effect onGgt inoculum in second year of pasture or crop. Unknown soil and environmental factors in the field precluded a simple relationship between grass level in pasture and subsequent level ofGgt inoculum, and where such a relationship did occur (2,4-D amine+propyzamide treatment) it appeared to be shortlived.     

Seasonal variations in larval biomass and biochemical composition of brown shrimp, Crangon crangon (Decapoda, Caridea), at hatching

The “brown shrimp”, Crangon crangon (Linnaeus 1758), is a benthic key species in the North Sea ecosystem, supporting an intense commercial fishery. Its reproductive pattern is characterized by a continuous spawning season from mid-winter to early autumn. During this extended period, C. crangon shows significant seasonal variations in egg size and embryonic biomass, which may influence larval quality at hatching. In the present study, we quantified seasonal changes in dry weight (W) and chemical composition (CHN, protein and lipid) of newly hatched larvae of C. crangon. Our data revealed significant variations, with maximum biomass values at the beginning of the hatching season (February–March), a decrease throughout spring (April–May) and a minimum in summer (June–September). While all absolute values of biomass and biochemical constituents per larva showed highly significant differences between months (P < 0.001), CHN, protein and lipid concentrations (expressed as percentage values of dry weight) showed only marginally significant differences (P < 0.05). According to generalized additive models (GAM), key variables of embryonic development exerted significant effects on larval condition at hatching: The larval carbon content (C) was positively correlated with embryonic carbon content shortly after egg-laying (r ² = 0.60; P < 0.001) and negatively with the average incubation temperature during the period of embryonic development (r ² = 0.35; P < 0.001). Additionally, water temperature (r ² = 0.57; P < 0.001) and food availability (phytoplankton C; r ² = 0.39; P < 0.001) at the time of hatching were negatively correlated with larval C content at hatching. In conclusion, “winter larvae” hatching from larger “winter eggs” showed higher initial values of biomass compared to “summer larvae” originating from smaller “summer eggs”. This indicates carry-over effects persisting from the embryonic to the larval phase. Since “winter larvae” are more likely exposed to poor nutritional conditions, intraspecific variability in larval biomass at hatching is interpreted as part of an adaptive reproductive strategy compensating for strong seasonality in plankton production and transitory periods of larval food limitation.     

Suppressive soil against Sclerotinia sclerotiorum as a source of potential biocontrol agents: selection and evaluation of Clonostachys rosea BAFC1646

The fungal diversity structures of soils that are suppressive and non-suppressive to Sclerotinia sclerotiorum were characterised and screened for fungal strains antagonistic to the S. sclerotiorum pathogen. Soil suppressiveness was associated with a particular fungal diversity structure. Principal component analysis showed that antagonism by fungal species in suppressive soils was associated with the occurrence of Fusarium oxysporum, Fusarium solani, Talaromyces flavus var. flavus and Clonostachys rosea f. rosea. In particular, C. rosea f. rosea occurred exclusively in suppressive soil samples, suggesting that this morpho-species plays an important role in suppression of S. sclerotiorum diseases. One strain of C. rosea f. rosea (BAFC1646) was selected for further experiments. Dual-culture assays confirmed the antagonistic behaviour of C. rosea f. rosea BAFC1646 against three different S. sclerotiorum strains. Antifungal activity was corroborated by diffusion assays with metabolite extracts. Greenhouse assays with soybean plants showed that the selected C. rosea f. rosea strain reduced the percentage of dead plants when co-inoculated with S. sclerotiorum. In addition, inclusion of C. rosea f. rosea alone increased shoot lengths significantly. In this work, we established the involvement of fungal species in soil suppressiveness and in further assays confirmed that C. rosea f. rosea BAFC1646 exhibits a bioprotective effect against S. sclerotiorum in soybean plants.     

The effect of moxidectin 0,1% vs ivermectin 0,08% on milk production in sheep naturally infected by gastrointestinal nematodes

Background  

Gastrointestinal nematode (GIN) infection is one of the main constraints to sheep production both in temperate and tropical countries. Economic losses caused by GIN are related to decreased production, treatment costs and even animal death. The present paper was aimed at assessing the anthelmintic efficacy (based on faecal egg count reduction) of moxidectin and ivermectin both admistered per os at dose rate of 0.2 mg/Kg body weight and the benefit of anthelmintic treatments on milk production in a commercial dairy sheep farms in central Italy whose animals were naturally infected by GIN.     

Morphological defenses induced in situ by the invertebrate predator Chaoborus : comparison of responses between Daphnia pulex and D. rosea

The presence of plankton predators may induce altered morphology in their potential prey. To date, the mechanism of induction and adaptive value of such defensive responses have been examined in the laboratory. This study investigated the morphological defense structures induced by the invertebrate predator Chaoborus in two coexisting Daphnia species, D. pulex and D. rosea, in the field. In Piscivore Lake (Gr?fenhain, Germany), continuous and intense biomanipulation had led to near elimination of planktivorous fish and greatly increased abundances of Chaoborus (up to >10 larvae l^–1). Here, the density of Chaoborus was manipulated within the lake by an enclosure/exclosure setup and resulting morphological responses of Daphnia spp. were investigated in situ. Three replicate enclosures (4.6 m³) contained no Chaoborus (predator exclusion bags), whereas Chaoborus entered three others at ambient densities (predator enclosures). In both species of Daphnia, formation of neckteeth and elongation of the tail spine were recorded in the predator enclosures, but not in the predator exclusion treatments. Additionally, D. rosea responded to predator inclusion with an increase of the size at first reproduction. Despite the induced defense structures, the presence of Chaoborus caused increased mortality of both Daphnia species. In addition, Chaoborus affected the coexistence of the two populations of Daphnia by causing higher relative mortality in D. rosea. Neckteeth formation was always more pronounced in D. pulex than in D. rosea of the same size. Neckteeth were induced specifically in vulnerably sized juvenile instars of D. pulex, but were not found in all vulnerable instars of D. rosea. In D. rosea, neckteeth were few or absent in the ephippial hatchlings, and neckteeth formation ceased before juveniles reached a body size outside the range that larger larval stages of Chaoborus could ingest. This study provides the first experimental demonstration in the field of the inducibility of morphological defense structures in Daphnia at ambient densities of Chaoborus larvae, and quantifies these in situ responses. This expands on earlier observations of a correlation between predator density in the field and the expression of neckteeth in Daphnia. The term ”maximum size for neckteeth formation” (MSNF) is defined as the limit in body size above which no production of neckteeth was evident. This limit was used to distinguish the size classes of Daphnia that show a sensitive response to Chaoborus kairomone. This new term may be used for further comparisons among species and among different types of predator-induced responses as well as for the evaluation of the adaptive value of defense structures. Received: 10 April 1999 / Accepted: 6 April 2000     

Pelagic food web interactions among benthic invertebrates and trout in mountain lakes

1. Benthic chironomid larvae and the amphipod Gammarus lacustris have been observed in the pelagic habitats of many mountain lakes. The main goal of this study was to determine if chironomid larvae and gammarids potentially affect predator–prey and nutrient dynamics in pelagic food webs of mountain lakes. 2. Eighty‐six mountain lakes were surveyed in Alberta and eastern British Columbia during the years 1965–1984, 1991–2004 and 2005–2007. Pelagic chironomid larvae were found in 86% of these lakes, and pelagic gammarids were found in 29% of lakes. Densities of pelagic chironomid larvae were 92% lower in lakes with pelagic gammarids and 76% lower in lakes with trout (P < 0.05). Intraguild predation of trout on gammarids appeared to reduce predation pressure on chironomid larvae. Gammarids consumed in vitro about 1 chironomid per gammarid per day or about 20% of their body mass in chironomid biomass per day. 3. Concentrations of total dissolved P and N, particulate C, and chlorophyll‐a increased with increasing densities of pelagic gammarids and chironomid larvae in situ (R² = 0.14 ± 0.19 SD, P < 0.1) and in vitro (P < 0.001). 4. Our findings suggest that gammarids and chironomid larvae are linked as predators and prey in pelagic food webs, possibly stimulating phytoplankton abundance via nutrient release.     

Photoperiodic and temperature effects on the intensity of larval diapause in Sesamia nonagrioides

Abstract. The intensity of larval diapause in Sesamia nonagrioides Lef (Lepidoptera: Noctuidae) was investigated under laboratory conditions. Newly hatched larvae were exposed to different stationary photoperiods (from LD 7 : 17 h to LD 14 : 10 h), at a constant temperature of 25 °C. Diapause incidence was higher when larvae were exposed to daylengths shorter than the critical value (LD 12 : 12 h), whereas the within‐treatment variation in the larval period appeared to be significantly correlated with the photoperiod applied. The incidences of diapause and the duration of larval development were also measured after exposing larvae to short photoperiods (LD 8 : 16 h, LD 10 : 14 h or LD 12 : 12 h) in combination with various temperatures (20, 22.5 or 25 °C). Although an increase in the incidence of diapause appeared with the lowering of the temperature, no statistical differences were observed in the time needed for pupation within the photoperiodic treatments at the temperatures of 20 and 22.5 °C. Furthermore, when diapausing larvae were transferred to the long photoperiod of LD 16 : 8 h, they immediately proceeded to pupation, regardless of the photoperiod or the temperature to which they had been previously exposed, indicating that there were no differences in the intensity of diapause. Photoperiodic changes from LD 10 : 14 h to LD 12 : 12 h or to LD 14 : 10 h at different larval ages reduced the intensity of diapause with (a) early age of transfer and (b) increase of daylength. By contrast, when larvae were transferred from the long photoperiod of LD 14 : 10 h to shorter, such as LD 10 : 14 h or LD 12 : 12 h, a small increase in the intensity of diapause with the shortening of the daylength was apparent. These results support the hypothesis that insects may compare the duration of the photoperiod and could classify them as either longer or shorter in relation to the critical value.     

A Transformation-induced Alteration of Cellular Membranes in Crown Gall Tumor Cells

Phospholipids were utilized as a membrane marker to test for transformation-induced alteration of cellular membranes of cultured crown gall cells of Vinca rosea L. Fully transformed cells contained less than half the amount of phospholipids (7.8 micrograms lipid P per gram fresh weight) of normal V. rosea cells (21.4 micrograms lipid P per gram fresh weight). The normal V. rosea callus cells were not significantly different (P > 0.05) in phospholipid content from partially transformed crown gall cells (20.7 micrograms lipid P per gram fresh weight). Stimulation to rapid growth of the partially transformed cells by adding higher concentrations of inorganic salts and auxin did not significantly alter their phospholipid content (23.1 micrograms lipid P per gram fresh weight). These findings suggest that the transformation process is directly responsible for an alteration of the cellular membranes and that the membrane alteration cannot be attributed to secondary effects associated with the rapid growth of these neoplastic cells.     

Temperature influences the performance and effectiveness of field and laboratory strains of the ichneumonid parasitoid, <Emphasis Type="Italic">Campoletis chlorideae</Emphasis>

To understand the influence of temperature on host–parasitoid interactions as a consequence of climatic change, we studied development, survival, and fecundity of field and laboratory strains of the Helicoverpa armigera larval endoparasitoid, Campoletis chlorideae at five different temperatures under laboratory conditions. Post-embryonic development period and degree-days required for completing the life cycle by both the strains decreased by 2.5 and 1.5 folds at 27°C compared to 18°C. Post embryonic development period showed a negative (r = −0.99, P < 0.001) and the development rate a positive (r = 0.99, P < 0.001) association with an increase in temperature. However, no parasitoid larvae survived in H. armigera larvae reared at 12 and 35°C after parasitization, suggesting that temperatures ≥35°C as a result of global warming will be lethal for development and survival of immature stages of C. chlorideae. Adult longevity was negatively associated (r = −0.91 to −0.96, P < 0.001) with temperatures between 12 and 35°C. The parasitoid adults stored at 12°C survived for longer period and exhibited higher fecundity than those kept at 27°C, but the efficiency of parasitism and adult emergence were quite low. Sex ratio of the progeny at 12°C was highly male-biased than the insects kept at 27°C. Laboratory strain of the parasitoid exhibited better survival, and the adults lived longer than the field strain at 18°C than at 27°C. Therefore, C. chlorideae adults stored at 18°C could be used for parasitism, while the immature stages should be reared at 27°C for mass production of the parasitoid for biological control of H. armigera.     

Comparison of raloxifene and atorvastatin effects on serum lipids composition of healthy post-menopausal women

The aim of this study was to evaluate the effects of the selective oestrogen receptor modulator, raloxifene, and those of statin, atorvastatin, in reducing the cardiovascular risks associated with the post-menopausal status. A detailed study of serum lipid concentrations was performed in four groups of post-menopausal women receiving either placebo, raloxifene or atorvastatin alone or their combination for the period of three months.Group A (raloxifene) showed significant decrease in total cholesterol levels (P < 0.05) and an increase in phospholipids concentration (P < 0.05), followed by a marked reduction in low-density lipoprotein cholesterol (LDL-C) levels (P < 0.01) and ApoB amounts (P < 0.001). Additionally, ApoA-I concentration was significantly increased (P < 0.01).Group B (atorvastatin) presented decreased cholesterol (P < 0.05) and triglycerides levels (P < 0.01), followed by elevated high-density lipoprotein cholesterol (HDL-C) concentration (P < 0.05) and low LDL-C amounts (P < 0.001). ApoA-I was significantly increased (P < 0.001) whereas ApoB was reduced (P < 0.001).The combined treatment in Group C (raloxifene and atorvastatin) showed significant changes in the majority of serum lipids. In particular, total cholesterol was reduced (P < 0.001), as well as triglycerides (P < 0.001) levels. Phospholipids were raised (P < 0.01) whereas LDL-C was reduced (P < 0.001) as was ApoB (P < 0.001). Furthermore, ApoA-I was elevated (P < 0.001). A further attempt to evaluate each treatment group was performed and the significance of these results is discussed. (Mol Cell Biochem 261: 71–75, 2004)     

Feeding habits of larval Mirogrex terraesanctae (Steinitz, 1952) in Lake Kinneret (Israel) I. Field study

Analysis of the food composition, distribution, lengths and dry weights of larval bleak, Mirogrex terraesanctae (Steinitz, 1952) in Lake Kinneret, Israel was conducted. Indices of electivity of ingested prey organisms were also determined. Larvae were surveyed throughout the 1989–90 reproductive seasons (Dec–Mar). Small larvae (<10.28 mm SL) preferentially select prey items smaller than 180 µ. Dominant zooplankters in the larval guts included: Bosmina longirostris, Ceriodaphnia reticulata and C. rigaudi, Asplanchna priodonita, Synchaeta oblonga, S. pectinata and juvenile cyclopoid copepods. Diaphanosoma brachiurum was rarely found (instars only) and no adult copepods were observed in larval guts. Some larvae specimens were found with up to 500 undigested (intact) accidentally ingested cells of Peridinium spp. per gut.     

Diapause induction as an interplay between seasonal token stimuli,and modifying and directly limiting factors: hibernation in Chymomyza costata

Larvae of wild type (WT) strain of Chymomyza costata Zetterstedt (Diptera: Drosophilidae) enter diapause (stop developing) in response to short‐day signal at a constant 18 °C, whereas larvae of a non‐photoperiodic‐diapause (NPD) strain do not respond to photoperiodic signalling and continue in larval development irrespective of daylength. The present study shows that WT larvae also respond reliably to thermoperiodic signalling (daily cycles of temperature) under constant darkness, whereas the NPD larvae do not, suggesting that the pathways transducing the environmental token stimuli (photoperiod and thermoperiod) onto the diapause developmental programme might merge functionally in the central biological clock system known to be mutated in NPD strain. Temperature and larval population density modify the output of token stimuli signalling. High temperatures (>24 °C) tend to avert, whereas low temperatures (<18 °C), especially in combination with constant darkness, stimulate diapause induction in WT strain. Overcrowding (>200 larvae per 5 g of larval diet) lengthens the duration of larval development and induces a ‘diapause‐like’ developmental arrest of relatively weak intensity in up to 60% of larvae of both strains. At high temperatures (>30 °C), all WT larvae continue direct development but subsequently die during the pupal stage. Low temperature exposure (<12 °C) causes quiescence in the majority of the larvae of both strains. Starvation blocks development and causes mortality when applied in larvae younger than day 3 of the third instar. Older larvae survive starvation and their photoperiodically‐induced developmental pre‐programming is not affected. Collectively, the results show that diapause induction in C. costata is a result of various interacting effects of multiple environmental factors.     

Selective and frequency dependent predation of aquatic mosquito predator Diplonychus indicus Venkatesan & Rao (Hemiptera: Belostomatidae) on immature stages of three mosquito species

Frequency dependent mosquito larval size (II and IV instars) and species selection by the water bug Diplonychus indicus against three mosquito species Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi was studied in the laboratory. The different frequencies used for each species selection were 20:30:50, 30:50:20, 50:20:30, 25:35:40, 35:40:25 and 40:25:35 of fourth instars of the respective three prey species. All nymphal water bugs (I–V instars) selected IV instar mosquito larvae and the mean proportion of late (larger) larvae eaten by the predator instars was significantly higher than the mean proportion of early (smaller) larvae eaten (F= 2.28; P < 0.001). In all six ratios used to determine the frequency dependent mosquito species selection, all the stages of the water bug selected Ae. aegypti over the other two species (F= 452.43; P < 0.001). The mean number of mosquito larvae eaten increased as its density increased based on various ratios of larvae offered. The study indicated that the predatory efficiency of D. indicus was high when Ae. aegypti was offered as prey, suggesting the utility of this mosquito predator in the control of dengue vectors.     

A Judgment on Postmortem Aging in Longissimus Dorsi Based on a Peptide Substrate Library

We attempted to develop a method to determine easily and effectively the degree of postmortem aging of pork longissimus dorsi (LD) by measuring the activity of proteases in the LD using fluorogenic peptide substrates. LD was used to measure the change with time in the protease activity detected with these substrates. Determining the variations within the LD muscles, strong positive correlations were found between changes in hardness and fluorescence intensities against Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P<0.005), and strong negative correlations were found between changes in total amounts of free amino acids and Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P<0.001). Negative correlations were also observed between changes in the amounts of free Ala, Arg, Lys, Leu, Met, Phe, and Tyr and the fluorescence intensities against Ala, Arg, Lys, Leu, Met, Phe, and Tyr-MCA respectively (P<0.001).     

Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC₅₀) values of the embryos and larvae were 1.71 and 0.22 mg l⁻¹ for copper and 2.57 and 2.72 mg l⁻¹ for zinc, respectively, whereas the MB LC₅₀ after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l⁻¹, respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l⁻¹ for embryos and 0.03, 0.03 and 1.78 mg l⁻¹ for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l⁻¹, respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l⁻¹ led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.