Genome survey sequencing of red swamp crayfish <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

Red swamp crayfish, Procambarus clarkii, presently is an important aquatic commercial species in China. The crayfish is a hot area of research focus, and its genetic improvement is quite urgent for the crayfish aquaculture in China. However, the knowledge of its genomic landscape is limited. In this study, a survey of P. clarkii genome was investigated based on Illumina’s Solexa sequencing platform. Meanwhile, its genome size was estimated using flow cytometry. Interestingly, the genome size estimated is about 8.50 Gb by flow cytometry and 1.86 Gb with genome survey sequencing. Based on the assembled genome sequences, total of 136,962 genes and 152,268 exons were predicted, and the predicted genes ranged from 150 to 12,807 bp in length. The survey sequences could help accelerate the progress of gene discovery involved in genetic diversity and evolutionary analysis, even though it could not successfully applied for estimation of P. clarkii genome size.     

Tolerance of acute hypercapnic acidosis by the European eel (<Emphasis Type="Italic">Anguilla anguilla</Emphasis>)

European eels ( Anguilla anguilla) were exposed sequentially to partial pressures of CO(2) in the water ( PwCO(2)) of 5, 10, 20, 40, 60 then 80 mm Hg (equivalent to 0.66-10.5 kPa), for 30 min at each level. This caused a profound drop in arterial plasma pH, from 7.9 to below 7.2, an increase in arterial PCO(2) from 3.0 mm Hg to 44 mm Hg, and a progressive decline in arterial blood O(2) content (caO(2)) from 10.0% to 1.97% volume. Gill ventilation rate increased significantly at water PwCO(2)s of 10, 20 and 40 mm Hg, followed by a decline at PwCO(2)s of 60 and 80 mm Hg, due to periodic breathing. Mean opercular pressure amplitude increased steadily throughout hypercapnic exposure and was significantly elevated at a PwCO(2) of 80 mm Hg. Hypercapnia caused a tachycardia between PwCO(2)s of 5 mmHg and 10 mm Hg, followed by a progressive decline in heart rate. Cardiac output (CO) remained unchanged throughout, as a consequence of a significant increase in stroke volume at PwCO(2)s of 40, 60 and 80 mm Hg. The eels maintained O(2) uptake at routine normocapnic levels throughout hypercapnic exposure. A comparison of the rates of blood O(2) delivery (calculated from CO and caO(2)) against O(2) consumption at PwCO(2)s of 60 mm Hg and 80 mm Hg indicated that a portion of O(2) uptake was due to cutaneous respiration. Thus, the European eel's exceptional tolerance of acute hypercapnia is probably a consequence of the tolerance of its heart to acidosis and hypoxia, and a contribution to O(2) uptake from cutaneous respiration.     

Light preferences of red swamp crayfish (<Emphasis Type="Italic">Procambarus clarkii</Emphasis>)

Light intensity preferences of Procambarus clarkii (Girard, 1852) were evaluated in an indoor fiber glass trough, consisting of five equal length (4 m) zones with light intensities varying from dark to 1,000 lux (L), situated at the ‘Las Vegas del Guadiana’ fish farm in Extremadura (Spain). Four trials were performed, with different light zones set in three at sections of the trough, and the fourth providing a control with one light density (300 L) throughout the trough. Whereas the highest intensity (1,000 L) was preferred in trials 1 and 2, the dimmest condition (D) was favored in trial 3. Crayfish showed a strong preference to both outside ends of the trough, and the “end effect” of the trough on position of the crayfish was found to be stronger than the impact of light intensity.     

Comparison of trophic function between the globally invasive crayfishes <Emphasis Type="Italic">Pacifastacus leniusculus</Emphasis> and <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

Impacts of invasive species may manifest most strongly if these organisms are highly distinct functionally from the native species they often replace. Yet, should we expect functional differences between native and invasive species of generalist organisms like freshwater crayfish? Some existing evidence has pointed to native and invasive crayfish species as ecologically equivalent. We contribute to this literature by comparing the trophic niches of the globally invasive crayfishes Pacifastacus leniusculus and Procambarus clarkii, by applying carbon and nitrogen stable isotope analyses to replicated allopatric (alone) and sympatric (together) lake populations in western Washington State, USA, where P. clarkii has been recently introduced and P. leniusculus is presumed native. Our study corrected for potential inherent differences in lake food webs as a consequence of lake abiotic or biotic characteristics using random effects in linear mixed effects models. We found that although overall trophic niche size or area of these species was not significantly different, P. leniusculus was significantly higher in trophic position than P. clarkii when also accounting for the effects of body size, sex, and lakes as random effects. This pattern of increased trophic position of P. leniusculus over P. clarkii was conserved over time in one sympatric lake for which we had data over multiple years. Cumulatively, our findings point to trophic differences between the globally cosmopolitan crayfishes P. leniusculus and P. clarkii, particularly when accounting for the ways that ecosystem context can affect food web structure of communities and the trophic resources available to these consumers.     

The North American crayfish <Emphasis Type="Italic">Procambarus clarkii</Emphasis> is the carrier of the oomycete <Emphasis Type="Italic">Aphanomyces astaci</Emphasis> in Italy

Aphanomyces astaci (Saprolegniales, Oomycetes) is classified among the 100 world’s worst invasive species. This species is endemic to North America and has been introduced into Europe by imports of their hosts, the North American crayfish species. As a consequence, extensive mass mortalities involved several populations of the European crayfish. Here, we checked its occurrence in four Italian populations of Procambarus clarkii, the most widespread alien crayfish in Italy. Digital image analyses and image processing techniques were used to select micro-melanized areas in the subabdominal cuticle of 2–10 crayfish per population. All the selected areas tested positive for A. astaci ITS nrDNA specific primers; moreover, the obtained sequences clearly corresponded to A. astaci, thus revealing that P. clarkii is an active carrier of this oomycete in Italy. Decisions are to be urgently made to control the spread of both P. clarkii and A. astaci for the conservation of the indigenous crayfish biodiversity.     

Biological control of broad mites (<Emphasis Type="Italic">Polyphagotarsonemus latus</Emphasis>) with the generalist predator <Emphasis Type="Italic">Amblyseius swirskii</Emphasis>

The broad mite is a serious pest of a variety of crops worldwide. Several phytoseiid mites have been described to control these mites. However, broad mites are still one of the major pest problems on greenhouse pepper in South-eastern Spain. The generalist predatory mite A. swirskii is widely used against other pests of pepper plants such as thrips and whiteflies, the latter being a vector of broad mites. We assessed the potential of A. swirskii to control broad mites. The oviposition rate of A. swirskii on a diet of broad mites was lower than on a diet of pollen, but higher than oviposition in the absence of food. Population-dynamical experiments with A. swirskii on single sweet pepper plants in a greenhouse compartment showed successful control of broad mites.     

Role of the olfactory pathway in agonistic behavior of crayfish, <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

Crayfish establish social dominance hierarchies through agonistic interactions, and these hierarchies are maintained through assessment of social status. Chemical signals influence several aspects of fighting behavior, but the specific chemosensory sensilla involved in detecting these signals in crayfish are unknown. The goal of our study was to examine the importance of aesthetasc sensilla—olfactory sensors on the antennules of decapod crustaceans—in regulating changes in fighting behavior in crayfish, Procambarus clarkii, over the course of repeated pairings. We selectively ablated aesthetascs from pairs of crayfish after the first day of trials and compared the behavior of these ablated animals to that of pairs of intact controls. Results show that unablated crayfish significantly decreased the number and duration of fights over repeated pairings, whereas crayfish lacking aesthetascs continued to engage in similar amounts of fighting across all three trial days. This difference shows that aesthetascs regulate fighting behavior in P. clarkii.     

Photopigment gene expression and rhabdom formation in the crayfish (<Emphasis Type="Italic">Procambarus clarkii</Emphasis>)

This study examines the expression of the photopigment gene in the developing retina of the freshwater crayfish Procambarus clarkii(Crustacea, Malacostraca, Decapoda). Both sense and anti-sense RNA probes were used for in situ hybridization (ISH) of whole embryos collected at various stages during development. A characteristic of retinal development is the formation of screening pigment in the retinular cells of the retinal ommatidia. This pigmentation is seen as a band that begins at the lateral side of the retinal field and progresses medially. At hatching the retina is approximately 50% pigmented. ISH of whole embryos shows that expression of the photopigment gene by the retinular cells correlates with the extent of the screening pigment band in the retina and with the presence of rhabdoms within the ommatidia. Sections taken through embryos after being hybridized indicate that staining is localized in the cytoplasm of the retinular cells and in the axonal region below the basement membrane. No staining reaction was seen in the rhabdoms of older ommatidia. ISH staining was also seen at the anterior midline of the protocerebrum where extraretinal photoreceptors have been reported. The data presented here show a close correlation of opsin expression within the retinular cells of the ommatidia and the formation of the very early rhabdoms, similar to Drosophila. The results will be discussed in relation to recent studies in Drosophila that suggest rhodopsin plays a role in effecting the organization of the terminal web-like cytoskeleton at the base of the developing rhabdom microvilli.     

<Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis>: a most successful invasive tick species in West-Africa

The cattle tick Rhipicephalus (Boophilus) microplus is known to be a highly reproductive and efficient vector of Babesia bovis, two characters which make this tick a threat to livestock keeping in many continents. The authors identified this tick in Ivory Coast, West Africa, in 2007, and hypothesized the spread to be minimal, as this tick was not observed in previous years. To determine the extent of its distribution and to a lesser extent the possible impact of the tick on the livelihoods of Ivorian smallholders, a cross-sectional survey was carried out in the Abidjan and Agboville Departments of Ivory Coast, in April 2008. The results of the study reveal that the newly introduced tick has almost completely displaced all indigenous Rhipicephalus (Boophilus) species in the study area and gave rise to unsuccessful tick control, inappropriate pesticide use, loss of milk production and even increased mortality in dairy cattle.     

Dietary effects on fatty acid composition in muscle tissue of juvenile European eel, <Emphasis Type="Italic">Anguilla anguilla</Emphasis> (L.)

The role of intracontinental migration patterns of European eel (Anguilla anguilla) receives more and more recognition in both ecological studies of the European eel and possible management measures, but small-scale patterns proved to be challenging to study. We experimentally investigated the suitability of fatty acid trophic markers to elucidate the utilization of feeding habitats. Eight groups of juvenile European eels were fed on eight different diets in a freshwater recirculation system at 20°C for 56 days. Three groups were fed on freshwater diets (Rutilus rutilus, Chironomidae larvae, and Gammarus pulex) and four groups were reared on diets of a marine origin (Clupea harengus, Crangon crangon, Mysis spec., and Euphausia superba) and one on commercial pellets used in eel aquaculture. Fatty acid composition (FAC) of diets differed significantly with habitat. FAC of eel muscle tissue seemed to be rather insensitive to fatty acids supplied with diet, but the general pattern of lower n3:n6 and EPA:ARA ratios in freshwater prey organisms could be traced in the respective eels. Multivariate statistics of the fatty acid composition of the eels resulted in two distinct groups representing freshwater and marine treatments. Results further indicate the capability of selectively restraining certain fatty acids in eel, as e.g. the n3:n6 ratio in all treatments was <4, regardless of dietary n3:n6. In future studies on wild eel, these measures can be used to elucidate the utilization of feeding habitats of individual European eel.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Evaluation of the toxicity of <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis> extract

In this study, the methanol extract of Arthrospira (Spirulina) platensis was examined for acute and subchronic toxicities. The extract did not produce any sign of toxicity within 7 days after feeding it at a single high dose of 6 g kg⁻¹ body weight to female and male Swiss mice. For the subchronic toxicity test, the extract at doses of 6, 12, and 24 mg kg⁻¹ body weight was orally administered to six male and six female Wistar rats daily for 12 weeks. Throughout the study period, we did not observe any abnormalities on behavior, food and water intakes, and health status among the treated animals. The hematology and clinical chemistry parameters of treated groups did not significantly differ from those of the controls in both sexes. Postmortem examination of the test groups also showed no abnormalities in both gross and histological findings. These results thus suggest that the methanol extract of A. platensis did not cause acute or subchronic toxicity in our experimental animals.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

First record of the tick <Emphasis Type="Italic">Ixodes</Emphasis> (<Emphasis Type="Italic">Pholeoixodes</Emphasis>) <Emphasis Type="Italic">kaiseri</Emphasis> in Turkey

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

<Emphasis Type="Italic">Sex combs reduced</Emphasis> (<Emphasis Type="Italic">Scr</Emphasis>) regulatory region of <Emphasis Type="Italic">Drosophila</Emphasis> revisited

The Hox gene Sex combs reduced (Scr) is responsible for the differentiation of the labial and prothoracic segments in Drosophila. Scr is expressed in several specific tissues throughout embryonic development, following a complex path that must be coordinated by an equally complex regulatory region. Although some cis-regulatory modules (CRMs) have been identified in the Scr regulatory region (~75 kb), there has been no detailed and systematic study of the distinct regulatory elements present within this region. In this study, the Scr regulatory region was revisited with the aim of filling this gap. We focused on the identification of Initiator elements (IEs) that bind segmentation factors, Polycomb response elements (PREs) that are recognized by the Polycomb and Trithorax complexes, as well as insulators and tethering elements. To this end, we summarized all currently available information, mainly obtained from high throughput ChIP data projects. In addition, a bioinformatic analysis based on the evolutionary conservation of regulatory sequences using the software MOTEVO was performed to identify IE and PRE candidates in the Scr region. The results obtained by this combined strategy are largely consistent with the CRMs previously identified in the Scr region and help to: (i) delimit them more accurately, (ii) subdivide two of them into different independent elements, (iii) identify a new CRM, (iv) identify the composition of their binding sites and (v) better define some of their characteristics. These positive results indicate that an approach that integrates functional and bioinformatic data might be useful to characterize other regulatory regions.     

Colonization of freshwater habitats by an introduced crayfish, <Emphasis Type="Italic">Procambarus clarkii</Emphasis>, in Southwest Iberian Peninsula

The introduction of some crustacean species has produced alterations of freshwater environments and declines of native species worldwide. The red swamp crayfish, Procambarus clarkii Girard, was introduced in the Southwest Iberian Peninsula in the 1970’s, producing severe impacts on rice agriculture and on native biota such as macrophytes, gastropods, native crayfish and amphibians. We studied the distribution of P. clarkii in two areas of SW Iberian Peninsula: the Sado River basin (SW Portugal), an area colonized by this species around 1990, and the Parque Natural del Entorno de Doñana (SW Spain), colonized soon after its introduction in the Iberian Peninsula, in the 1970’s. Our main goal was to determine which factors limit crayfish distribution, which could help to identify the most effective management practices to contain its spread. Procambarus clarkii was found in most types of water bodies, including small and shallow ones. Distance to a crayfish source was the single predictor variable explaining crayfish occurrence in most types of habitats and in both areas. The only exception was for the Sado permanent stream points, where crayfish presence was negatively affected by an interaction between elevation and flow velocity. Other habitat characteristics have apparently little or no importance for its successful colonization. Moreover, this study indicated that overland dispersal is apparently a frequent phenomenon in this species. Our findings can be used to determine which habitats are most likely to be colonized by the crayfish and to develop practical measures which may limit its spread and minimize its impacts.