The ability of rumen ciliates, Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum, to use the murein saccharides

Murein polysaccharides may contribute to a considerable part of the dry matter of bacterial cells. Their utilization by protozoa inhabiting the rumen is, however, poorly recognized. The objective of this study was to examine the ability of three species of ciliates, i.e., Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum of digest, and ferment these saccharides. The cultivation experiments showed that the enrichment of growth medium with bacterial cell wall β-glycans increased the ciliate number (p?<?0.05). A statistically significant increase (p?<?0.01) was followed by a continuous decrease (p?<?0.01) in the percentage of individuals containing β-glycans particles after 4- and 24-h incubation of ciliates with this substrate, respectively. The enzymatic experiments confirmed the ability of the examined protozoa to digest murein. E. caudatum exhibited the highest activity (8.2 unit (U)/mg protein per min), and E. maggii, the lowest (3.0 U/mg protein per min). The production rates of volatile fatty acids by starved and fed ciliate species were 0.7 and 1.6 (E. caudatum)?pmol/ciliate cell per h, 30.5 and 42.5 (E. maggii)?pmol/ciliate cell per h, and 8.3 and 19.2 (D. affine)?pmol/ciliate cell per h (p?<?0.05).     

Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of −30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.     

The macronuclear genome of anaerobic ciliate Entodinium caudatum reveals its biological features adapted to the distinct rumen environment

Entodinium caudatum is an anaerobic binucleated ciliate representing the most dominant protozoal species in the rumen. However, its biological features are largely unknown due to the inability to establish an axenic culture. In this study, we primally sequenced its macronucleus (MAC) genome to aid the understanding of its metabolism, physiology, ecology. We isolated the MAC of E. caudatum strain MZG-1 and sequenced the MAC genome using Illumina MiSeq, MinION, and PacBio RSII systems. De novo assembly of the MiSeq sequence reads followed with subsequent scaffolding with MinION and PacBio reads resulted in a draft MAC genome about 117 Mbp. A large number of carbohydrate-active enzymes were likely acquired through horizontal gene transfer. About 8.74% of the E. caudatum predicted proteome was predicted as proteases. The MAC genome of E. caudatum will help better understand its important roles in rumen carbohydrate metabolism, and interaction with other members of the rumen microbiome.     

The 3' Untranslated Region of Messages in the Rumen Protozoan Entodinium caudatum

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.     

In vitro methane formation and carbohydrate fermentation by rumen microbes as influenced by selected rumen ciliate species

Ciliate protozoa contribute to ruminal digestion and emission of the greenhouse gas methane. Individual species of ciliates co-cultured with mixed prokaryote populations were hypothesized to utilize carbohydrate types differently. In an in vitro batch culture experiment, 0.6 g of pure cellulose or xylan was incubated for 24 h in 40-mL cultures of Entodinium caudatum, Epidinium ecaudatum, and Eudiplodinium maggii with accompanying prokaryotes. Irrespective of ciliate species, gas formation (mL) and short-chain fatty acids (SCFA) concentrations (mmol L^?1) were higher with xylan (71; 156) than with cellulose (52; 105). Methane did not differ (7.9% of total gas). The SCFA profiles resulting from fermentation of the carbohydrates were similar before and after removing the ciliates from the mixed microbial population. However, absolute methane production (mL 24 h^?1) was lower by 50% on average after removing E. caudatum and E. maggii. Methanogen copies were less without E. maggii, but not without E. ecaudatum. Within 3 weeks part of this difference was compensated. Butyrate proportion was higher in cultures with E. maggii and E. ecaudatum than with E. caudatum and only when fermenting xylan. In conclusion, the three ciliate species partly differed in their response to carbohydrate type and in supporting methane formation.     

Suitability of different media for in vitro cultivation of the ruminal protozoa species Entodinium caudatum, Eudiplodinium maggii, and Epidinium ecaudatum

Three protozoal cultivation media were tested to determine the medium which best facilitated growth and viability of key B-type ciliates isolated from the sheep rumen. Entodinium caudatum and Eudiplodinium maggii were grown anaerobically in 50-ml flasks for 32 days in Caudatum-type (C), Kisidayova (K) or Dehority (M) medium. On day 32, in media K and M, E. caudatum cell counts were high with 5.6 × 10³ and 7.8 × 10³ mL⁻¹, respectively, and the proportion of dead cells was low with 0.6 and 1.4%, respectively. E. maggii concentrations when grown in medium M and C were 2.7 × 10³ and 2.4 × 10³ mL⁻¹, respectively, with 3.9 and 14.1% dead cells. Medium M, which favoured growth of both protozoa species, was tested again and Epidinium ecaudatum was included. Protozoa were grown for a 4-month period and samples were taken in the last two months on days 1, 7, 35 and 57. Average cell concentrations were 10.0, 0.8 and 0.5 × 10³ mL⁻¹ for E. caudatum, E. maggii, and E. ecaudatum, respectively. In conclusion, medium M would appear to be the best choice for cultivating these three species in one medium.     

Rumen ciliates of domestic cattle (Bos taurus taurus) in Kastamonu,Turkey, with the description of a new species

Species composition and distribution of ciliates were investigated in the rumen contents of 25 domestic cattle (Bos taurus taurus L.) living in Kastamonu, Turkey. Forty-seven species and 37 morphotypes representing 15 genera were identified. Of them, a new species of Ostracodinium was recognized and described as Ostracodinium anatolicum n. sp. This new species has two caudal lobes. The dorsal lobe is small and rounded and the ventral lobe is triangular shaped and bent toward the dorsal side like a thick hook. Furthermore, the anterior end of the macronucleus (1/5 of the length) is bent toward the left like a hook. The density of rumen ciliates in cattle was 96.8 ± 43.3 × 10⁴ cells mL⁻¹ and the mean number of ciliate species per host was 14.2 ± 4.4. Entodinium longinucleatum, E. nanellum, E. simulans and Isotricha prostoma were the most abundant species, each with a prevalence of 88%. Entodinium chatterjeei, E. bifidum m. monospinosum, Hsiungia triciliata, Oligoisotricha bubali, Ostracodinium dogieli, O. mammosum and O. munham are new host records for cattle from Turkey.     

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.     

Nutritional characterization of Mucuna pruiriens: 2. In vitro ruminal fluid fermentability of Mucuna pruriens,Mucunal-dopa and soybean meal incubated with or without l-dopa

Three in vitro experiments were conducted to determine the rumen fermentability of Mucuna (M) pruriens (24 g 3,4-dihydroxy-l-phenylalanine (l-dopa)/kg dry matter (DM) and soybean meal treated with (SBD) or without (SB) 138 g l-dopa/kg DM). Additional objectives were to determine if l-dopa inhibits rumen fermentation, and if ruminal microbes can adapt to l-dopa or M. In Experiment 1, ground (1 mm) substrates were incubated in triplicate at 38 °C in 9 ml nutrient media and 1 ml rumen fluid in a series of six, 48 h, consecutive batch cultures. The first culture was inoculated with rumen fluid from two donor cows. Subsequent cultures were inoculated with fluid (1 ml) from the previous culture. The DM digestibility (DMD, 616 g/kg vs. 540 g/kg; P<0.01) and gas production (51.7 ml/g vs. 44.2 ml/g DM; P<0.05) were higher from fermentation of M versus SB but similar for SB and SBD (540 g/kg vs. 554 g/kg and 44.2 ml/g DM vs. 43.5 ml/g DM, respectively). The slopes of the relationships between DMD (g/kg) or gas production (ml/g DM) and fermentation period were not reduced by fermenting M (−0.014 DMD slope; 2.28 gas production slope) or SBD (−0.014 DMD slope; 0.459 gas production slope), instead of SB (−0.002 DMD slope; 1.039 gas production slope), indicating microbial adaptation to M and SBD. Total volatile fatty acid concentration (VFA; 53.7, 54.9 and 54.9 mmol/l) and molar proportions of VFA were similar among substrates. Gas production kinetics of M versus SB (Experiment 2), and SB versus SBD (Experiment 3) were also measured after substrates were incubated in triplicate in buffered rumen fluid for 24 h using a non-linear exponential model to fit the data. Residual l-dopa was measured after separate fermentation of substrates in triplicate for 0, 4, 8, 16 and 24 h. Fermentation of M versus SB produced more (P<0.05) gas (250 ml/g vs. 100 ml/g DM) and total VFA (203 mmol/l vs. 180 mmol/l) and a lower (P<0.05) acetate:propionate ratio (1.35 vs. 1.87; P<0.05). Adding l-dopa to SB increased (P<0.01) gas production (92 ml/g DM vs. 200 ml/g DM), and total VFA concentration (132 mmol/l vs. 188 mmol/l), but reduced (P<0.05) gas production rate (0.08 ml/h vs. 0.05 ml/h). The concentration of l-dopa in fermented M and SBD decreased by 53 and 47%, respectively during fermentation. In conclusion, M was more fermented than SB and degradation of l-dopa during ruminal fermentation and microbial adaptation to l-dopa were confirmed. Adding l-dopa to SB did not impair ruminal fermentation.     

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum , a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium , was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum . Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.     

Tyrosinase activity of Greyia flanaganii (Bolus) constituents

Hyper-pigmentation of the skin is a common problem that is prevalent in middle aged and elderly people. It is caused by over production of melanin. Tyrosinase is known to be the key enzyme in melanin production. Ethanolic extract of Greyia flanaganii leaves showed significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 32.62 μg/ml. The total extract was further investigated for its toxicity and effect on melanin production by melanocytes cells, and showed significant inhibition (P < 0.05) (20%) of melanin production at 6.25 μg/ml and low levels of cytotoxicity (IC₅₀ < 400 μg/ml). The amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (EC₅₀) by the total ethanolic extract was found to be 22.01 μg/ml. The effect of G. flanaganii against acne causing bacteria, Propionibacterium acnes, was investigated using microdilution assay. The MIC of the extract of G. flanaganii was found to be 250 μg/ml. Bioassay-guided fractionation led to the isolation of (3S)-4-hydroxyphenethyl 3-hydroxy-5-phenylpentanoate (1), 2′,4′,6′-trihydroxydihydrochalcone (2), 2′,6′,4-trihydroxy-4′-methoxydihydrochalcone (3), 2′,6′-dihydroxy-4′-methoxydihydrochalcone (4), 5,7-dihydroxyflavanone [(2S)-pinocembrin] (5), 2′,6′-dihydroxy-4′,4-dimethoxy dihydrochalcone (6) and (2R,3R)-3,5,7-trihydroxy-3-O-acetylflavanone (7). The isolated compounds were tested for their antioxidant, cytotoxicity, tyrosinase inhibition and antibacterial activities. Compound 2 exhibited significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 69.15 μM. The isolated compounds showed low toxicity of the cells with reduction of melanin content of the cells. All compounds tested showed good radical scavenging activity. These data indicates that G. flanaganii extract and its isolated phenolic constituents could be possible skin lightening agents.     

Dodonaea viscosa var. angustifolia leaf: New source of polysaccharide and its anti-oxidant activity

Ultrasonic technology was applied for polysaccharide extraction from the leaves of Dodonaea viscosa and response surface methodology (RSM) was used to optimize the effects of processing parameters on polysaccharide extraction yield. Three independent variables were extraction time (X₁), extraction temperature (X₂) and ultrasonic power (X₃), respectively. The statistical analysis indicated the independent variables (X₁, X₂, X₃), the quadratic terms (X₁₁ and X₃₃) and the interaction terms (X₁X₂, X₁X₃, X₂X₃) had significant effects on the yield of polysaccharides (P < 0.05). The optimal extraction conditions of D. viscosa leaf were determined as follows: extraction time 50.54 min, extraction temperature 85 °C and ultrasonic power 400 W. Under these conditions, the experimental yield of polysaccharides was 9.455 ± 0.24%, which was agreed closely with the predicted value (9.398%). The evaluation of anti-oxidant activity suggested that the polysaccharide exhibited significant protection against DPPH and hydroxyl radicals and could be explored as a nutraceutical agent.     

Bacterial Ingestion by the Rumen Ciliates Entodinium and Diplodinium

SYNOPSIS. In cattle fed a high-starch diet, species of Entodinium and Diplodinium ingested associated ruminal bacteria. Stained preparations of diluted rumen contents showed Entodinium caudatum, E. minimum, E. dubardi , (syn. E. simplex ), E. longinucleatum, E. bursa, E. nanellum, E. exiguum , and E. vorax contained gram-positive diplococci. Starch grains with adherent gram-positive diplococci were observed within Entodinium spp. Diplodinium ecaudatum forma ecaudatum, D. ecaudatum forma caudatum, D. neglectum and an unidentified species of Diplodinium also ingested ruminal diplococci. Bacteria were isolated from mixed species of Entodinium by washing and culturing the protozoa in a starch feed-extract agar medium. The strains isolated from the ciliates were gram-positive diplococci, 0.8 times 1–1.5 μm, which attached themselves to starch granules and were able to digest the starch. Conclusive evidence of bacterial ingestion by the oligotrichs was obtained by providing the bacterial cultures to Entodinium species ( E. dubardi and E. minimum ) which had been starved 24 hr. Gram-stained preparations showed the ciliates readily ingested the bacteria. The amylolytic cocci utilized by Entodinium spp. were identified as Streptococcus bovis.     

Rumen Bacterial and Protozoal Responses to Insecticide Substrates

Insecticides containing organophosphate, chlorinated hydrocarbon, and carbamate were tested with bovine ruminal ingesta fractions. Rumen bacteria exposed to insecticide levels of 0 to 500 ppm in rumen fluid for 4 hr were inoculated into rumen fluid-starch feed extract medium. No apparent significant bacterial count inhibitions were noted. Also, when insecticides were used as carbon sources at concentrations of 500 ppm in carbohydrate-limited media, no increases in bacterial counts were indicated. Warburg manometric data showed that paraffin oil-Triton X-155 preparations of dimethoate, Diazinon, lindane, Thiodan and Sevin stimulated gas production in holotrich protozoa. Entodinium simplex, an oligotrich, produced less gas with insecticide substrates per unit of dry weight than did an Isotricha sp. Rumen bacteria and plant debris fractions from ruminal ingesta provided with insecticides did not give increased manometric responses over the endogenous control vessels. Washed suspensions of I. intestinalis produced volatile fatty acids in excess of the endogenous suspensions when provided insecticide substrates. Thiodan dissimilation by I. intestinalis was followed colorimetrically with 15% loss in substrate in 1 hr of incubation at 39 C. Diazinon-C¹⁴ substrate uptake was demonstrated with suspensions of E. simplex and I. intestinalis. Rumen ciliates are suggested as a possible means for screening out useful insecticides susceptible to microbial dissimilation for use on forage and other cattle-feed crops.     

Effect of harvest date and nitrogen fertilization rate on the nutritive value of amaranth forage (Amaranthus hypochondriacus)

This study was conducted to assess effects of harvest date (i.e., 40 and 60 d after planting) and N fertilization rate (i.e., 120, 180, 240 kg N/ha) on the nutritive value of amaranth forage (Amaranthus hypochondriacus) using a factorial experiment with a randomized complete block design. The content of dry matter (DM), crude protein (CP), true protein (TP), ether extract (EE), water soluble carbohydrates (WSC), ash-free neutral detergent fiber (NDFom), ash-free acid detergent fiber (ADFom), lignin(sa), ash, Ca, P, Na, K, oxalic acid and nitrate were determined. Soluble CP (SP) and protein fractions non-protein N (A), true protein rapidly degraded in the rumen (B₁), true protein degraded in the rumen at a moderate rate (B₂), true protein associated with the cell wall and slowly degraded in the rumen (B₃) and acid detergent insoluble CP (C) were measured according to the Cornell Net Carbohydrate and Protein System. In vitro gas production (IVGP), OM disappearance (OMD) and NDFom disappearance (NDFD) were determined using a gas production technique. Results showed that the later harvest date increased (P<0.05) DM, EE, WSC, NDFom, ADFom, lignin(sa), B₃ and C; while CP, TP, ash, Ca, P, K, SP, A, B₁, B₂, nitrate, total and soluble oxalic acid, IVGP, b (i.e., gas production from the insoluble fermentable fractions at 120 h), c (i.e., rate of gas production during incubation), OMD and NDFD decreased (P<0.05). With increasing N fertilization rate, CP, TP, EE, P, nitrate, oxalic acid, SP, A, b, OMD and NDFD increased (P<0.05), however B₂ declined (P<0.05). Increasing N fertilization increased yield, CP concentration and nutrient digestibility. At 40 d after planting use of amaranth forage as a ruminant feed is limited due to its high nitrate content. However, at 60 d, although a depression in digestibility and CP content occurred, this forage has the potential as a ruminant feed due to the much lower nitrate levels.     

Antifeedant activity of ethanolic extract from Flourensia oolepis and isolation of pinocembrin as its active principle compound

The ethanolic extract from Flourensia oolepis aerial parts showed strong antifeedant activity against the pest larvae, Epilachna paenulata, with an antifeedant index (AI%) of 99.1% at 100 μg/cm². Based on chromatographic fractionation of the extract, guided by bioassays on E. paenulata, the flavanone pinocembrin (1) was isolated as the most active principle. In a choice assay, compound 1 showed strong antifeedant activity against E. paenulata, Xanthogaleruca luteola and Spodoptera frugiperda with an AI% of 90, 94 and 91% (p < 0.01) respectively, at 50 μg/cm². The dosages necessary for 50% feeding inhibition of the insects (ED₅₀) were 7.98, 6.13 and 8.86 μg/cm², respectively. The feeding inhibitory activity of 1 against E. paenulata was compared with the activity of other structurally related flavonoids like naringenin, which was inactive up to 100 μg/cm², catechin which was nearly 6 times less active than 1, and quercetin which was equally active as 1. The effect of these on the feeding behavior of E. paenulata was also studied.     

Genotyping of isolates of Clostridium perfringens from vaccinated and unvaccinated sheep

Enterotoxaemia vaccine is a polyvalence vaccine from different types of Clostridium perfringens, and C. septicum toxins that prevents various diseases, the most important one being enterotoxaemia in sheep. The aim of the present study was to evaluate the enterotoxaemia vaccine effects in reducing isolates of intestinal clostridia genus specifically C. perfringens. Sheep dung samples were randomly collected from 10 places in Kerman, Iran. The samples were taken from 90 vaccinated Kermani sheep against enterotoxaemia and 50 unvaccinated sheep of same age from flocks with similar management. Following processing and culture of the samples, colonies were identified applying morphological, gram stain and biochemical tests. Using these tests C. perfringens were isolated from 27 out of 50 unvaccinated sheep (54.0%) and from 2 out of 90 vaccinated sheep (2.2%). All of the clostridia isolates were analyzed by multiplex PCR. Genotyping of 2 strains isolated from the vaccinated sheep indicated that these strains were type D, while the strains isolated from the unvaccinated sheep were types A, B, C and D; 14.8% (4 out of 27), 22.2% (6 out of 27), 40.7% (11 out of 27) and 22.2% (6 out of 27), respectively. However, no isolate containing the iota gene (type E) was detected. Vaccination against enterotoxaemia had a significant effect (P < 0.01) on reducing C. perfringens isolates. Occurrence of the disease in the vaccinated and unvaccinated groups was 3.3% and 64.0% (P < 0.01), respectively.     

Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites

The repellent activity of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), and the main iridoid compounds (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone, was assessed against (i) major Afro-tropical pathogen vector mosquitoes, i.e. the malaria mosquito, Anopheles gambiae s.s. and the Southern house mosquito, Culex quinquefasciatus, using a World Health Organisation (WHO)-approved topical application bioassay (ii) the brown ear tick, Rhipicephalus appendiculatus, using a climbing repellency assay, and (iii) the red poultry mite, Dermanyssus gallinae, using field trapping experiments. Gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS) analysis of two N. cataria chemotypes (A and B) used in the repellency assays showed that (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone were present in different proportions, with one of the oils (from chemotype A) being dominated by the (4aS,7S,7aR) isomer (91.95% by GC), and the other oil (from chemotype B) containing the two (4aS,7S,7aR) and (4aS,7S,7aS) isomers in 16.98% and 69.83% (by GC), respectively. The sesquiterpene hydrocarbon (E)-(1R,9S)-caryophyllene was identified as the only other major component in the oils (8.05% and 13.19% by GC, respectively). Using the topical application bioassay, the oils showed high repellent activity (chemotype A RD₅₀ = 0.081 mg cm⁻² and chemotype B RD₅₀ = 0.091 mg cm⁻²) for An. gambiae comparable with the synthetic repellent DEET (RD₅₀ = 0.12 mg cm⁻²), whilst for Cx. quinquefasciatus, lower repellent activity was recorded (chemotype A RD₅₀ = 0.34 mg cm⁻² and chemotype B RD₅₀ = 0.074 mg cm⁻²). Further repellency testing against An. gambiae using the purified (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone isomers revealed overall lower repellent activity, compared to the chemotype A and B oils. Testing of binary mixtures of the (4aS,7S,7aR) and (4aS,7S,7aS) isomers across a range of ratios, but all at the same overall dose (0.1 mg), revealed not only a synergistic effect between the two, but also a surprising ratio-dependent effect, with lower activity for the pure isomers and equivalent or near-equivalent mixtures, but higher activity for non-equivalent ratios. Furthermore, a binary mixture of (4aS,7S,7aR) and (4aS,7S,7aS) isomers, in a ratio equivalent to that found in chemotype B oil, was less repellent than the oil itself, when tested at two doses equivalent to 0.1 and 0.01 mg chemotype B oil. The three-component blend including (E)-(1R,9S)-caryophyllene at the level found in chemotype B oil had the same activity as chemotype B oil. In a tick climbing repellency assay using R. appendiculatus, the oils showed high repellent activity comparable with data for other repellent essential oils (chemotype A RD₅₀ = 0.005 mg and chemotype B RD₅₀ = 0.0012 mg). In field trapping assays with D. gallinae, addition of the chemotype A and B oils, and a combination of the two, to traps pre-conditioned with D. gallinae, all resulted in a significant reduction of D. gallinae trap capture. In summary, these data suggest that although the nepetalactone isomers have the potential to be used in human and livestock protection against major pathogen vectors, intact, i.e. unfractionated, Nepeta spp. oils offer potentially greater protection, due to the presence of both nepetalactone isomers and other components such as (E)-(1R,9S)-caryophyllene.     

The effects of algae species and densities on the population growth of the marine rotifer, Colurella dicentra

Colurella dicentra clones isolated from bay water in the Mississippi Gulf Coast were cultured with artificial seawater. Experiments were conducted to determine the effects of six algae species (Nannochloropsis oculata, Tetraselmis chuii, Chaetoceros gracilis, Rhodomonas salina, Isochrysis galbana, and Prorocentrum micans), six C. gracilis densities, and six N. oculata densities (25,000, 50,000, 100,000, 250,000, 500,000, and 1,000,000 cells ml^− 1) on C. dicentra population growth. Algae type influenced rotifer production (p < 0.0001). C. gracilis treatment (9120 ± 3351SD) produced the highest number of rotifers followed by N. oculata (5760 ±2232SD). P. micans had the lowest number of rotifers, although not significantly different from numbers in T. chuii, R. salina, and I. galbana treatments (p > 0.05).The population growth rate (r) varied with algae species treatment. The highest values were recorded for C. gracilis treatment (0.22 to 0.26 d^− 1), followed by N. oculata (0.21 to 0.24 d^− 1), and the lowest for P. micans (− 0.19 to 0.14 d^− 1). C. gracilis and N. oculata densities had significant effects (p < 0.0001) on C. dicentra population growth. The highest rotifer production was recorded at a C. gracilis density of 100,000 cells ml^− 1, followed by 250,000 cells ml^− 1 and 50,000 cells ml^− 1. Algae densities of 500,000 cells ml^− 1 and above produced the lowest rotifer numbers. Population growth rate (r) varied with C. gracilis densities. The highest values were observed for C. gracilis concentrations of 100,000 cells ml^− 1 (0.17 to 0.19 d^− 1), and the lowest for concentrations of 500,000 cells ml^− 1 and above (− 0.19 to 0.09 d^− 1). The 100,000 cells ml^− 1N. oculata density gave the highest rotifer production followed by 50,000, 250,000, 25,000, and 500,000 cells ml^− 1. Algae densities of 1,000,000 cells ml^− 1 produced the lowest rotifer numbers. Population growth rate (r) varied with N. oculata densities, with the highest values obtained for algae densities of 100,000 cells ml^− 1 (0.35 to 0.40 d^− 1), and the lowest for concentrations of 1,000,000 cells ml^− 1 (0.05 to 0.012 d^− 1). This is the first report of C. dicentra in Mississippi Coastal waters, and perhaps the smallest marine rotifer species (93 by 49 μm) ever cultured successfully.     

Effect of Acanthocephala infection on the reproductive potential of crustacean intermediate hosts

The effect of a naturally acquired infection by three acanthocephalan parasites Dentitruncus truttae, Echinorhynchus truttae, and Polymorphus minutus on the reproductive potential of their intermediate host, Echinogammarus tibaldii (Amphipoda) from Lake Piediluco (Centre of Italy) was assessed. During May 2007, 1135 amphipods were collected from two different samplings and examined for larval helminths. Forty-five amphipods were infected and of those, 16 were infected with D. truttae (intensity = 1-3 larvae), 15 with E. truttae (intensity = 1-2 larvae), and 14 with P. minutus (intensity = 1 larva). The sex ratio was nearly 1:1 in all examined amphipods. One female infected with D. truttae contained six eggs in the brood pouch and another female infected with E. truttae contained five eggs. However, none of the eight female amphipods harbouring P. minutus larva contained eggs in their brood pouch. Uninfected females of the same size and body length as that of the infected females contained between 20 and 32 eggs. No acanthocephalan species were found to co-occur.