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.     

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.     

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.     

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.     

Biological control of invasive populations of crayfish: the European eel (<Emphasis Type="Italic">Anguilla anguilla</Emphasis>) as a predator of <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

The red swamp crayfish, Procambarus clarkii, is a paradigmatic invader of freshwater systems. Several attempts have been made to mitigate its multiple impacts but none was successful. Among the different methods proposed, the use of the European eel (Anguilla anguilla) as an indigenous predator is promising but the available information about its predatory ability on crayfish is to date scanty. To fill this gap in knowledge, we ran three experiments in wetlands and irrigation ditches in Italy. The first experiment, in the laboratory, was aimed at quantifying the extent of predation by eels on crayfish, the second, in enclosures, the size classes of crayfish mainly preyed and the possible effect of the eels on P. clarkii behaviour, and the third, in the field, its ability to effectively reduce crayfish populations. Results showed that eels prey on small-sized or soft crayfish, attacking them from the back; an indirect effect was to reduce crayfish trophic activity, which in turn might increases crayfish mortality due to starvation and decreases impact on the community. However, as shown in the field, the use of eels should be appropriately calibrated to the context of application. Taken together, our results show that eels might be used as a complement to the traditional trapping method. However, additional studies are necessary to understand the adequate number of eels to be introduced and to develop appropriate methods for quantifying such effects.     

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.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Prevalence of full-size <Emphasis Type="Italic">P</Emphasis> and <Emphasis Type="Italic">KP</Emphasis> elements in North American populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The P transposable element invaded the Drosophila melanogaster genome in the middle of the twentieth century, probably from D. willistoni in the Caribbean or southeastern North America. P elements then spread rapidly and became ubiquitous worldwide in wild populations of D. melanogaster by 1980. To study the dynamics and long-term fate of transposable genetic elements, we examined the molecular profile of genomic P elements and the phenotype in the P-M system of the current North American natural populations collected in 2001-2003. We found that full-size P and KP elements were the two major size classes of P elements present in the genomes of all populations ("FP + KP predominance") and that the P-related phenotypes had largely not changed since the 1980s. Both FP + KP predominance and phenotypic stability were also seen in other populations from other continents. As North American populations did not show many KP elements in earlier samples, we hypothesize that KP elements have spread and multiplied in the last 20 years in North America. We suggest that this may be due to a transpositional advantage of KP elements, rather than to a role in P-element regulation.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Proteomic approach: Identification of <Emphasis Type="Italic">Medicago truncatula</Emphasis> proteins induced in roots after infection with the pathogenic oomycete <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>

The legume root rot disease caused by the oomycete pathogen Aphanomyces euteiches is one major yield reducing factor in legume crop production. A comparative proteomic approach was carried out in order to identify proteins of the model legume Medicago truncatula which are regulated after an infection with A. euteiches. Several proteins were identified by two dimensional gel electrophoresis to be differentially expressed after pathogen challenge. Densitometric evaluation of expression values showed different regulation during the time-course analysed. Proteins regulated during the infection were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Among the differentially expressed proteins, two encoded putative cell wall proteins and two were designated as small heat shock proteins. Furthermore, an isoform of the chalcone-O-methyltransferase was found to be increased in infected roots. The majority of induced proteins belonged to the family of class 10 of pathogenesis related proteins (PR10). Previously, various PR10-like proteins have been shown to be regulated by general stress or abscisic acid (ABA). Therefore, these proteins were further investigated concerning their regulation in response to drought stress and exogenous ABA-application. Complex regulation patterns were identified: three of the A. euteiches-induced PR10-like proteins were also induced by exogenous ABA- but none of them is induced after drought stress. In contrast, three of these proteins are down-regulated by drought stress. Hence, the strong expression of different PR10-family members and their regulation profiles indicates that this set of proteins plays a major role during root adaptations to various stress conditions.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.