A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (≥75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation ≤60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (≥80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.     

Expression of the decapentaplegic ortholog in embryos of the onychophoran Euperipatoides rowelli

The gene decapentaplegic (dpp) and its homologs are essential for establishing the dorsoventral body axis in arthropods and vertebrates. However, the expression of dpp is not uniform among different arthropod groups. While this gene is expressed along the dorsal body region in insects, its expression occurs in a mesenchymal group of cells called cumulus in the early spider embryo. A cumulus-like structure has also been reported from centipedes, suggesting that it might be either an ancestral feature of arthropods or a derived feature (=synapomorphy) uniting the chelicerates and myriapods. To decide between these two alternatives, we analysed the expression patterns of a dpp ortholog in a representative of one of the closest arthropod relatives, the onychophoran Euperipatoides rowelli. Our data revealed unique expression patterns in the early mesoderm anlagen of the antennal segment and in the dorsal and ventral extra-embryonic tissue, suggesting a divergent role of dpp in these tissues in Onychophora. In contrast, the expression of dpp in the dorsal limb portions resembles that in arthropods, except that it occurs in the mesoderm rather than in the ectoderm of the onychophoran limbs. A careful inspection of embryos of E. rowelli revealed no cumulus-like accumulation of dpp expressing cells at any developmental stage, suggesting that this feature is either a derived feature of chelicerates or a synapomorphy uniting the chelicerates and myriapods.     

Class I antigens of the bovine major histocompatibility system are weakly associated with variation in faecal worm egg counts in naturally infected cattle

Three bulls selected for high faecal worm egg counts and three bulls selected for low faecal worm egg counts were mated to Africander-Hereford cross cows. Faecal worm egg counts were taken on four occasions from the 132 offspring. Also, each animal was typed for 32 class I antigens of the bovine major histocompatibility system (BoLA). Least squares analysis of variance showed that line, sex and some of the antigens were associated with differences in worm egg output in the faeces. After adjusting for the effects of line and sex, cattle with antigen W9 had about twice as many worm eggs in their faeces as cattle without W9; cattle with antigen CA45 had about half the concentration of faecal worm eggs as cattle without CA45. However, the antigen associations were of borderline significance at the 5% level and more work in additional populations is necessary to confirm these associations.     

Expression of the decapentaplegic ortholog in embryos of the onychophoran Euperipatoides rowelli

The gene decapentaplegic (dpp) and its homologs are essential for establishing the dorsoventral body axis in arthropods and vertebrates. However, the expression of dpp is not uniform among different arthropod groups. While this gene is expressed along the dorsal body region in insects, its expression occurs in a mesenchymal group of cells called cumulus in the early spider embryo. A cumulus-like structure has also been reported from centipedes, suggesting that it might be either an ancestral feature of arthropods or a derived feature (=synapomorphy) uniting the chelicerates and myriapods. To decide between these two alternatives, we analysed the expression patterns of a dpp ortholog in a representative of one of the closest arthropod relatives, the onychophoran Euperipatoides rowelli. Our data revealed unique expression patterns in the early mesoderm anlagen of the antennal segment and in the dorsal and ventral extra-embryonic tissue, suggesting a divergent role of dpp in these tissues in Onychophora. In contrast, the expression of dpp in the dorsal limb portions resembles that in arthropods, except that it occurs in the mesoderm rather than in the ectoderm of the onychophoran limbs. A careful inspection of embryos of E. rowelli revealed no cumulus-like accumulation of dpp expressing cells at any developmental stage, suggesting that this feature is either a derived feature of chelicerates or a synapomorphy uniting the chelicerates and myriapods.     

Knot formation reduces water exchange by adult males of the hairworm,Paragordius varius (Nematomorpha: Gordioidea)

To examine how aggregation by hairworms may enhance survival in freshwater, we determined water balance characteristics of Paragordius varius in groups of different sizes. P. varius is hyperosmotic resulting in high body water content and functions down to one-half of water stores. Absence of a critical transition temperature implies a watertight, low-porosity cuticle. Aggregated worms lose water slowly, as a physiological consequence of reduced motor activity. The water balance strategy shifts from a reliance on high dehydration tolerance for isolated individuals, affording high water loss rates, to suppressed activation energy when aggregated, wherein blocking water gain is important when water loss is slower. Low water loss rate derives from stillness and aggregating that facilitate mating or anti-predator defense, rather than as a behavior to regulate water loss. Presence of hairworms in streams is an indicator of high-quality water that is necessary to maintain water balance.     

Automated and customizable quantitative image analysis of whole Caenorhabditis elegans germlines

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-content image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: (1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and (2) assessment of these individual nuclei based on their position within the germline. We show the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double-strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields using the C. elegans germline as a model system.     

Field investigation of pomegranate fruit worms in Taif and laboratory evaluation of Bacillus thuringiensis against Ectomyelois ceratoniae

Field observation during the period of pomegranate production (from May to October) in Taif showed that pomegranate fruits are attacked by two insects, Ectomyelois ceratoniae and Virachola livia. Survey results indicate that the larvae of Ectomyelois ceratoniae was more effective compared with the larvae of Virachola livia. Biological control tests were carried out with commercial Bacillus thuringiensis against the second and fourth instar larvae of Ectomyelois ceratoniae under laboratory conditions. Fourth instar larvae were the most sensitive to bacteria at different concentrations (ppm) compared with the second instar larvae. The survival resulting from higher concentrations was lowest. Larvae fed on dry grapes treated by Bt. appeared to be changed in body color to the white before death.     

The importance of measuring biotic and abiotic factors in the lower egg pocket to predict coho salmon egg survival

Based on results from simulated redds of coho salmon Oncorhynchus kisutch , the amount of fine sediment <0·5 mm in the lower half of the egg pocket, rather than the entire egg pocket of the redd, was a strong predictor of egg survival to hatching (  r ²  = 0·62). The relationship was much stronger than observed in other studies, which typically ignore egg pocket structure. Abundance of a fish egg-eating worm, Haplotaxis ichthyophagous , an oligochaete that may have been attracted to fine sediment and dead eggs in the egg pocket, was also associated with a decrease in egg survival. The worm, however, accounted for little of the variance in survival compared to fine sediment. Only 10% fine sediment (<0·5 mm) in the lower pocket was required to decrease survival from 100 to 5%. Other abiotic factors had weaker (gravel permeability) or non-existent (dissolved oxygen) correlations with survival.     

Studies on the involvement of prostaglandins and their precursors in the rejection of Nippostrongylus brasiliensis from the rat

In vitro incubation of 6-day Nippo-strongylus brasiliensis in the presence of PGE₁ at 1000 ng/ml and PGE₂ at 500–10,000 ng/ml of medium did not affect worm motility nor in vivo survival of worms implanted into the small intestine of recipient rats. The intraduodenal injection of 250 and 500 μg PGE₁ or PGE₂ did not lead to expulsion of worms from infected rats. An in vitro exposure to precursor fatty acids of PGE₁ and PGE₂, dihomo-γ-linolenic acid and arachidonic acid, respectively, at concentrations of 1000–15,000 ng/ml of medium also failed to inhibit worm motility and in vivo worm survival. These results are at variance with some earlier reports and do not suggest that prostaglandins are directly involved in the immune rejection of N. brasiliensis. No prostaglandins could be demonstrated in worm homogenates.     

Studies with Brugia pahangi. II. The effect of repeated infection on parasite levels in cats

Denham D. A., Ponnudurai T., Nelson G. S., Rogers Rosemary and Guy Frances 1972. Studies with Brugia pahangi—II. The effect of repeated infection on parasite levels in cats. International Journal for Parasitology2: 401–407. 21 cats were given a primary infection of 100–200 infective larvae of Brugia pahangi followed, some time later, by repeated challenge with 50 larvae per time at 10-day intervals. In most cats the microfilarial levels increased considerably but in a minority the levels remained the same as those seen in cats given only one infection. Adult worm recoveries were very much higher than after a single infection but after about 20 challenges there was no further increase in the number of worms establishing an infection. After a long series of challenge infections, the microfilarial counts of some cats suddenly fell and the blood became free of microfilariae.