Identification and expression profiling of Ceratitis capitata genes coding for β-hexosaminidases

The goal of this study was to identify the genes coding for β-N-acetylhexosaminidases in the Mediterranean fruit fly (medfly) Ceratitis capitata, one of the most destructive agricultural pests, belonging to the Tephritidae family, order Diptera. Two dimeric β-N-acetylhexosaminidases, HEXA and HEXB, have been recently identified on Drosophila sperm. These enzymes are involved in egg binding through interactions with complementary carbohydrates on the surface of the egg shell. Three genes, Hexosaminidase 1 (Hexo1), Hexosaminidase 2 (Hexo2) and fused lobes (fdl), encode for HEXA and HEXB subunits. The availability of C. capitata EST libraries derived from embryos and adult heads allowed us to identify three sequences homologous to the D. melanogaster Hexo1, Hexo2 and fdl genes. Here, we report the expression profile analysis of CcHexo1, CcHexo2 and Ccfdld in several tissues, organs and stages. Ccfdl expression was highest in heads of both sexes and in whole adult females. In the testis and ovary the three genes showed distinct spatial and temporal expression patterns. All the mRNAs were detectable in early stages of spermatogenesis; CcHexo2 and Ccfdl were also expressed in early elongating spermatid cysts. All three genes are expressed in the ovarian nurse cells. CcHexo1 and Ccfdl are stage specific, since they have been observed in stages 12 and 13 during oocyte growth, when programmed cell death occurs in nurse cells. The expression pattern of the three genes in medfly gonads suggests that, as their Drosophila counterparts, they may encode for proteins involved in gametogenesis and fertilization.     

Is polyandry a common event among wild populations of the pest Ceratitis capitata?

In many insect species, females can mate more than once and store sperm from more than one male. An assessment and understanding of polyandry in the field can be important for pest species with a high colonization potential, such as the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), which is also highly polyphagous and among the most destructive agricultural insects. The use of polymorphic microsatellite markers, combined with different statistical approaches, provides evidence that polyandry occurs in two C. capitata natural populations, one population from the Greek island of Chios and one population from Rehovot, in Israel. The observed different level of polyandry is discussed in relation to the genetic diversity, seasonality, and demography of the two populations. When polyandry is present, paternity analysis also indicates that one male, presumably the last, tends to sire most of the progeny. Polyandry and paternity skew may have important implications for the evolution of the species, in terms of maintenance of the genetic variability. Moreover, these aspects of the mating behavior, i.e., remating frequency and paternity skew, may locally affect the sterile insect technique, the most commonly applied control strategy against C. capitata.     

Travel medicine and general practice: a suitable case for audit?

Travel medicine becomes more important with the continual expansion of international travel and the increased popularity of exotic holiday destinations. In the United Kingdom general practitioners provide the bulk of travel health advice and immunisation and there is growing interest in providing these services. While their armamentarium has been expanded with attractive but expensive new vaccines, the need for health service advice has never been more vital, with the risks of HIV infection and drug resistant malaria. Advantages of a general practice based travel medicine service include maintaining continuity of care for the patient, but a disadvantage might be that the general practitioner sees too few patients to acquire enough skill in the subject. Furthermore, there may be a conflict of interest between time devoted to the "vaccination ritual" and giving health advice. Overall there seems to be a case for both audit and support by the health authorities.     

Tyrosine-4-O-β-glucoside in the mediterranean fruit-fly Ceratitis capitata

Tyrosine-4-O-β-glucoside was isolated from 5-day-old larvae of Ceratitis capitata, and its structure was established by biochemical and physical techniques. Tyrosine glucoside is the major metabolic product derived from tyrosine during the last larval stage of the fruit-fly.     

Two-stage case–control designs for rare genetic variants

The search for the association of rare genetic variants with common diseases is of high interest, yet challenging because of cost considerations. We present an efficient two-stage design that uses diseased cases to first screen for rare variants at stage-1. If too few cases are found to carry any variants, the study stops. Otherwise, the selected variants are screened at stage-2 in a larger set of cases and controls, and the frequency of variants is compared between cases and controls by an exact test that corrects for the stage-1 ascertainment. Simulations show that our new method provides conservative Type-I error rates, similar to the conservative aspect of Fisher’s exact test. We show that the probability of stopping at stage-1 increases with a smaller number of cases screened at stage-1, a larger stage-1 continuation threshold, or a smaller carrier probability. Our simulations also show how these factors impact the power at stage-2. To balance stopping early when there are few variant carriers versus continuation to stage-2 when the variants have a reasonable effect size on the phenotype, we provide guidance on designing an optimal study that minimizes the expected sample size when the null hypothesis is true, yet achieves the desired power.     

Feeding habits of Ceratitis capitata (Diptera: Tephritidae): Can larvae recognize a nutritionally effective diet?

Larvae of Ceratitis capitata were tested for preference for different diets based on the ingredients of the standard diet used to rear flies in the laboratory. The nutritional value of these diets was also determined. The results showed that larvae are able to recognize the diets that are best from a nutritional viewpoint. When chemically defined diets were tested, larvae always preferred those containing the largest amount of protein, except for older larvae, which preferred protein-free diets. In a threshold study, larvae were able to recognize as little as 0.1 g yeast/100 ml diet and 1.5 casein/100 ml diet.     

Infectious disease — a genetic view

Genetic analysis of resistance to infectious disease reveals many important cues that have led to new insights into the interaction between pathogen and host. This knowledge might help with a better prognosis for diseases, and to the development of novel therapeutics. This review focuses on genes and loci that control susceptibility to diseases with an important epidemiologic impact, such as AIDS, hepatitis B, gastritis and peptic ulcer, tuberculosis, leprosy, malaria, schistosomiasis and leishmaniasis. New perspectives for the integration of human and mouse genetics that contribute greatly to our understanding of regulatory mechanisms in health and disease, are also discussed.     

Preparative centrifugation — a new method for preparing pollen concentrates suitable for radiocarbon dating by AMS

Methods of preparative centrifugation eliminate many of the difficulties involved in preparing pollen concentrates from deposits rich in resistant organic material. Density centrifugation for the separation of pollen from a gyttja sample rich in resistant organic matter was tested. Combining centrifugations in two CsCl solutions, one of higher density and one of lower density than pollen, a pure pollen fraction was successfully prepared. Data on the isodensity and sedimentation rate of fossilized recent pollen from twelve tree taxa are also presented, and the potential for separating a single taxon from pollen assemblages is demonstrated.     

Protoplast fusion — a tool for genetic manipulation and breeding in industrial microorganisms

Protoplasts can be isolated from microbial cells by enzymatic digestion of the cell wall, in the presence of an osmotic stabilizer. Such protoplasts can be induced to fuse in the presence of agents such as the poly (ethylene glycols). When suitably selected auxotrophic strains are used, the fusion products can be recovered by selection on the basis of nutritional complementation. Cultivation of the protoplasts on a hypertonic growth medium induces regeneration of new cell wall material and their subsequent reversion to the normal cell form of the organism. The protoplast fusion technique has been applied sucessfully to both bacterial and fungal systems leading to the recovery of recombinant progeny. In the fungi, the recovery of non-parental segregants from inter-species crosses has also been demonstrated. In assessing the value of the fusion technique, caution may be necessary at this stage in its application to genetic mapping in bacteria. The behaviour of protoplasts, especially with respect to reversion, could be an additional factor that operates during selection, distorting recombination frequencies. However, the fusion technique, in providing a mechanism by which genetic recombination can be readily achieved, should be of great potential in empirical breeding and strain improvement. These aspects are reviewed.     

Morphological and genetic divergence of intralacustrine stickleback morphs in Iceland: a case for selective differentiation?

The evolutionary processes involved in population divergence and local adaptation are poorly understood. Theory predicts that divergence of adjacent populations is possible but depends on several factors including gene flow, divergent selection, population size and the number of genes involved in divergence and their distribution on the genome. We analyse variation in neutral markers, markers linked to putative quantitative trait loci and morphological traits in a recent (<10000 years) zone of primary divergence between stickleback morphs in Lake Thingvallavatn, Iceland. Environmental factors, especially predation, are clearly implicated in reducing gene flow between morphs. There is continuous morphological and genetic variation between habitats with a zone centre similar to secondary contact zones. Individual microsatellite loci are implicated as being linked to adaptive variation by direct tests as well as by differences in cline shape. Patterns of linkage disequilibria indicate that the morphs have diverged at several loci. This divergence shows parallels and differences with the well-studied limnetic-benthic stickleback morphs, both in phenotypic divergence and at the genomic level.     

Vitellogenin levels in mussel hemolymph—a suitable biomarker for the exposure to estrogens?

Increased vitellogenin (vtg) levels in the blood of male fish are frequently used as an indicator of estrogenic exposure. Similar responses are expected for mussels, where the concentration of vtg-like proteins has been reported to depend on estrogens. To verify the role of hemolymph during vitellogenesis of mussels, the saltwater mussel Mytilus edulis and the freshwater mussel Anodonta cygnea were exposed to 17beta-estradiol (E2) and wastewater treatment plant effluents, known for their estrogenic potential. Gel electrophoresis did not reveal any significant induction (or repression) of plasma proteins compared to control plasma. Our results do not support the hypothesis that mussel hemolymph is a carrier of estrogen-dependent major egg-yolk precursors (vtg-like proteins). However, additional information on a 35+/-2-kDa hemolymph protein, previously reported to bind heavy metals, was obtained by high-resolution two-dimensional electrophoresis. It was resolved in a cluster of single proteins with properties that match the characteristics of a previously reported histidine-rich glycoprotein.     

Human microbiome: a genetic bazaar for microbes?

A recent study suggests that lateral gene transfer has been particularly intense among human-associated microbes. What can this tell us about our relationship with our internal microbial world?     

Glycosylase mediated polymorphism detection (GMPD)—a novel process for genetic analysis

A process for mutation and polymorphism detection is described here that offers significant advances over current mutation detection systems and that has the potential to significantly enhance molecular genetic analysis of human disease. This novel process is referred to as glycosylase mediated polymorphism detection (GMPD) and exploits the use of highly specific DNA glycosylase enzymes to excise substrate bases incorporated into amplified DNA. Action of the glycosylase leaves the DNA with one or more specific abasic sites which can be cleaved by enzymatic or chemical means. The GMPD process permits detection of polymorphisms and mutations using fragment size analysis or solid phase formats. GMPD is particularly suitable for genotyping of single nucleotide polymorphism (SNP) based markers and also permits efficient scanning of genes for unknown polymorphisms and mutations.     

Are blue-green algae a suitable food for zooplankton? An overview

One of the reasons suggested to explain the dominance of blue-greens in eutrophic lakes is that they are not used as food by zooplankton; and even when ingested, they are poorly utilized.

An increase in herbivores might be the expected result of biomanipulation of the aquatic food chain. This attempt at controlling the algae population is, however, destined to fail if zooplankton do not also utilize blue-greens as food. In this respect, a series of in-lake experimental results indicates that after the food chain has been biomanipulated, there is a decrease in blue-green density in periods when there is an increase in herbivores. Is this only an accidental result or are the two facts interrelated; in other words, can the decrease in the density of blue-greens be attributed to the increased use of them by zooplankton herbivores?

The suitability of blue-greens as food for zooplankton has been widely investigated by many authors with contrasting and inconclusive results. Two main factors seem to play important role in determining their suitability as food: the biochemical properties of the different species, or even different strains of the same species; and the shape and size of the colonies.

In particular, biochemical properties can result in toxic effects on zooplankton, while size and shape may strongly interfere with filtering, thus reducing the possibility of gathering food.

     

Synthesis and characterization of a fluorescent probe for α-tocopherol suitable for fluorescence microscopy

Previously prepared fluorescent derivatives of α-tocopherol have shown tremendous utility in both in vitro exploration of the mechanism of ligand transfer by the α-tocopherol transfer protein (α-TTP) and the intracellular transport of α-tocopherol in cells and tissues. We report here the synthesis of a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) containing α-tocopherol analog having extended conjugation with an alkenyl thiophene group that extends the absorption and emission maxima to longer wavelengths (λ_ex = 571 nm and λ_em = 583 nm). The final fluorophore thienyl-ene-BODIPY-α-tocopherol, 2, binds to recombinant human α-TTP with a K_d = 8.7 ± 1.1 nM and is a suitable probe for monitoring the secretion of α-tocopherol from cultured Mcf7#189 cells.     

In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by the impaired functioning of ciliated cells. Its diagnosis is based on the analysis of the structure and functioning of cilia present in the respiratory epithelium (RE) of the patient. Abnormalities of cilia caused by hereditary mutations closely resemble and often overlap with defects induced by the environmental factors. As a result, proper diagnosis of PCD is difficult and may require repeated sampling of patients’ tissue, which is not always possible. The culturing of differentiated cells and tissues derived from the human RE seems to be the best way to diagnose PCD, to study genotype–phenotype relations of genes involved in ciliary dysfunction, as well as other aspects related to the functioning of the RE. In this review, different methods of culturing differentiated cells and tissues derived from the human RE, along with their potential and limitations, are summarized. Several considerations with respect to the factors influencing the process of in vitro differentiation (cell-to-cell interactions, medium composition, cell-support substrate) are also discussed.