Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.     

Clinical evaluation of clonidine added to lidocaine solution for subarachnoid analgesia in sheep

Clonidine (CL) is a alpha2-adrenergic agonist that produces analgesia in animals and humans by a non-opiate alpha2-adrenergic action in the spinal cord dorsal horn. The objective of this prospective randomized study was to investigate the clinical effects of CL/lidocaine (LD) combination administered by the subarachnoid route in sheep. Each sheep received each of three treatments, at no shorter than weekly intervals. Treatments consisted of 0.003 mg/kg CL, 1.2 mg/kg LD and a combination of CL (0.003 mg/kg) and LD (1.2 mg/kg) (CLLD). Subarachnoid injections were given in all animals between the last lumbar and first sacral vertebra. Heart rate (HR), arterial pressures, respiratory rate, rectal temperature, analgesia, sedation, and motor blockade were determined before drug administration (basal) and 5, 10, 15 and 30 min after drug administration, and at 30-min intervals until loss of analgesia occurred. The duration of analgesia after subarachnoid CLLD administration was 187 +/- 24 min (mean +/- SD), i.e. more than twice of that obtained with CL (99 +/- 19 min) or LD (55 +/- 4.4 min) alone. In all sheep, CL, administered either alone or with LD, induced moderate sedation. After subarachnoid administration of three treatments, all sheep had ataxia and subsequent sternal recumbency. The CL treatment causes decreases in blood pressure (diastolic arterial pressure and mean arterial pressure) and HR. Data suggest that the CLLD combination could be used subarachnoidally in sheep requiring prolonged surgery.     

Doubled-haploid versus single-seed descent and S1-family variation for testcross performance in a maize population

Progress made in the in situ gynogenesis technique since 1990 now allows production of a high number of maize (Zea mays L.) doubled-haploid (DH) lines. The aim of the study was to compare DH lines versus selfing lines for testcross performance. DH and single-seed descent (SSD) lines were produced from random S₁ progenies of a broad-base population. For grain yield, kernel moisture, plant height, ear height and leaf length, the three population means were similar. Except for kernel moisture, the genetic variance of DH lines was nearly twice as high as the genetic variance of S₁ families, as expected. On the other hand, genetic variance among SSD lines was only 1.5 times higher than the genetic variance of S₁ families. This lower variance could be due to a selection bias in the method of production of SSD lines. However, for all traits, heritability of SSD or DH lines was higher than heritability of S₁ families. Epistasis effects in DH progenies were not significant. The consequence was a high correlation between S₁ testcross progenies and DH or SSD testcross progenies, meaning that the S₁ testcross value can be used to select the best families from which DH lines will be extracted. As a whole, the observed variation in DH lines appeared to be more in accordance with the observed variation among S₁ families than with the observed variation among SSD lines.     

Unrecognized diversity in New Guinean crayfish species (Decapoda,Parastacidae): The evidence from molecular data

The phylogenetic relationships among imported ornamental crayfish belonging to the genus Cherax were inferred from a combined dataset of 3 mitochondrial genes (COI, 16S and 12S) and by comparison with available GenBank sequences of 14 Cherax species. Furthermore, the concordance of previously described species obtained from a wholesaler (Cherax boesemani, C. holthuisi and C. peknyi) with available GenBank sequences was verified based on COI with special respect to comparison with sequences assigned as Cherax species. Recently described species C. gherardiae, C. pulcher and C. subterigneus belong to the northern group of Cherax species. Comparison and analysis with other GenBank COI sequences show previously unreported diversity of New Guinean species, suggesting 5 putative new species. Surprisingly, species assigned to the subgenus Astaconephrops do not form a monophyletic clade; this subgenus should be reappraised relative to the purported typical morphological characteristic of the uncalcified patch on male chelae. Increasing importation of crayfish underscores the importance of accurate species identification. Use of basic molecular methods is a necessary requisite for documenting occurrence, abundance and population trends of target species. Consequently, it helps to support eventual conservation decision‐making by stakeholders.     

Interactions of Pratylenchus thornei and Fusarium oxysporum f. sp. ciceris on Chickpea

ABSTRACT Fusarium oxysporum f. sp. ciceris and the root-lesion nematode Pratylenchus thornei coinfect chickpeas in southern Spain. The influence of root infection by P. thornei on the reaction of Fusarium wilt-susceptible (CPS 1 and PV 61) and wilt-resistant (UC 27) chickpea cultivars to F. oxysporum f. sp. ciceris race 5 was investigated under controlled and field conditions. Severity of Fusarium wilt was not modified by coinfection of chickpeas by P. thornei and F. oxysporum f. sp. ciceris, in simultaneous or sequential inoculations with the pathogens. Root infection with five nematodes per cm(3) of soil and 5,000 chlamydospores per g of soil of the fungus resulted in significantly higher numbers of propagules of F. oxysporum f. sp. ciceris with the wilt-susceptible cultivar CPS 1, but not with the wilt-resistant one. However, infection with 10 nematodes per cm(3) of soil significantly increased root infection by F. oxysporum f. sp. ciceris in both cultivars, irrespective of fungal inoculum densities (250 to 2,000 chlamydospores per g of soil). Plant growth was significantly reduced by P. thornei infection on wilt-susceptible and wilt-resistant chickpeas in controlled and field conditions, except when shorter periods of incubation (45 days after inoculation) were used under controlled conditions. Severity of root necrosis was greater in wilt-susceptible and wilt-resistant cultivars when nematodes were present in the root, irrespective of length of incubation time (45 to 90 days), densities of nematodes (5 and 10 nematodes per cm(3) of soil), fungal inocula, and experimental conditions. Nematode reproduction on the wilt-susceptible cultivars, but not on the wilt-resistant one, was significantly increased by F. oxysporum f. sp. ciceris infections under controlled and field conditions.     

Alginate Microsphere Encapsulation of Drug-Loaded Nanoparticles: A Novel Strategy for Intraperitoneal Drug Delivery

Alginate hydrogels have been broadly investigated for use in medical applications due to their biocompatibility and the possibility to encapsulate cells, proteins, and drugs. In the treatment of peritoneal metastasis, rapid drug clearance from the peritoneal cavity is a major challenge. Aiming to delay drug absorption and reduce toxic side effects, cabazitaxel (CAB)-loaded poly(alkyl cyanoacrylate) (PACA) nanoparticles were encapsulated in alginate microspheres. The PACAlg alginate microspheres were synthesized by electrostatic droplet generation and the physicochemical properties, stability, drug release kinetics, and mesothelial cytotoxicity were analyzed before biodistribution and therapeutic efficacy were studied in mice. The 450 µm microspheres were stable at in vivo conditions for at least 21 days after intraperitoneal implantation in mice, and distributed evenly throughout the peritoneal cavity without aggregation or adhesion. The nanoparticles were stably retained in the alginate microspheres, and nanoparticle toxicity to mesothelial cells was reduced, while the therapeutic efficacy of free CAB was maintained or improved in vivo. Altogether, this work presents the alginate encapsulation of drug-loaded nanoparticles as a promising novel strategy for the treatment of peritoneal metastasis that can improve the therapeutic ratio between toxicity and therapeutic efficacy.     

Competitive and allelopathic interference between soybean crop and annual wormwood (Artemisia annua L.) under field conditions

Annual wormwood interference on soybean crop growth and yield may result from competition and allelopathy, which are modulated by crop management. Allelochemicals released by annual wormwood (e.g. artemisinin) may affect the crop directly or indirectly through the effect on the nitrogen fixing symbiont, Bradyrhizobium japonicum. The objectives were (i) to quantify the crop response (i.e. biomass production, nodulation and yield) to weed interference and (ii) to determinate the relative change of competition and allelopathy interferences, when a sublethal dose of herbicide is applied. Two split plot field experiments with three replications were used. The experiment involved a factorial combination of five weed–crop density (soybean/annual wormwood, plants m^?2) levels: D1, pure soybean, 40/0 plants m^?2; D2, 40/2 plants m^?2; D3, 40/4 plants m^?2 and D4, 40/8 plants m^?2, and D5, pure annual wormwood, 0/8 plants m^?2, two activated carbon (allelopathy) levels: C?, with activated carbon (reduced allelopathy) and C+, non activated carbon applied (with allelopathy) and two herbicide levels: H?, untreated and H+, treated with a sub-lethal dose of glyphosate. Activated carbon to adsorb allelochemicals (with and without activated carbon) and glyphosate application (with and no herbicide) were assigned to sub-plots. Increasing weed density did not affect crop biomass at flowering, but changed nodule number and soybean yield with a different pattern depending on carbon and herbicide treatment. Relative crop yield decreased with increasing relative weed biomass. This decrease was particularly drastic when allelopathy was reduced by activated carbon and without herbicide application. The maximum yield losses of 33% in 2006 and 17% in 2007 were observed with the highest weed density (8 plants m^?2). In contrast, without carbon (high allelopathy level), soybean yield remained stable within the explored range of annual wormwood biomass, despite the fact that weed biomass at high densities (D4) was high enough to generate competition. The lack of response to increasing weed density could be related to the indirect effect of allelochemicals interacting with soil microorganisms (i.e. B. japonicum) that positively affected the nodulation (e.g. larger nodules in 2006 and increased nodules biomass due to higher number of roots in 2007 at high densities). With herbicide application, soybean yield of both carbon treatments remained stable when biomass of annual wormwood increased. This research provided strong evidence in support of the existence of positive effect of allelopathic and competitive interactions between annual wormwood and soybean crop under field conditions that may be overridden under herbicide application.     

Characterization of the LH peak after short and long fixed‐time artificial insemination protocols in sheep raised in the tropics

The aim of this study was to evaluate the peak in luteinizing hormone (LH) and the pregnancy rate of sheep (Texel × Santa Inês) in the tropics using short‐ (6 days) and long‐term (12 days) progesterone protocols followed by artificial insemination (AI) both in and out of the breeding season. Experiment 1 was conducted within (IN) the breeding season (autumn, n = 36), and experiment 2 was conducted outside (OUT) of the breeding season (spring, n = 43). In each experiment, the sheep were divided into two groups (6 or 12 days) according to the duration of treatment with a single‐use progesterone release vaginal device (CIDR^®, Pfizer, São Paulo, SP, Brazil), and blood samples were collected from 10 animals per group every 4 hr to measure the LH and progesterone concentrations. In the spring, the characteristics of the LH peak did not differ between groups; but in the autumn, there were differences between groups at the beginning (G‐6 IN: 36.44 ± 5.46 hr; G‐12 IN: 26.57 ± 4.99 hr) and end of the LH peak (G‐6 IN: 46.22 ± 7.51 hr; G‐12 IN: 34.86 ± 8.86 hr). The results showed alterations in the LH peak during the breeding season only in the sheep undergoing the short‐term protocol.