RESUMEN
Babesia bovis, is a tick borne apicomplexan parasite responsible for important cattle losses globally. Babesia parasites have a complex life cycle including asexual replication in the mammalian host and sexual reproduction in the tick vector. Novel control strategies aimed at limiting transmission of the parasite are needed, but transmission blocking vaccine candidates remain undefined. Expression of HAP2 has been recognized as critical for the fertilization of parasites in the Babesia-related Plasmodium, and is a leading candidate for a transmission blocking vaccine against malaria. Hereby we identified the B. bovis hap2 gene and demonstrated that it is widely conserved and differentially transcribed during development within the tick midgut, but not by blood stage parasites. The hap2 gene was disrupted by transfecting B. bovis with a plasmid containing the flanking regions of the hap2 gene and the GPF-BSD gene under the control of the ef-1α-B promoter. Comparison of in vitro growth between a hap2-KO B. bovis clonal line and its parental wild type strain showed that HAP2 is not required for the development of B. bovis in erythrocytes. However, xanthurenic acid-in vitro induction experiments of sexual stages of parasites recovered after tick transmission resulted in surface expression of HAP2 exclusively in sexual stage induced parasites. In addition, hap2-KO parasites were not able to develop such sexual stages as defined both by morphology and by expression of the B. bovis sexual marker genes 6-Cys A and B. Together, the data strongly suggests that tick midgut stage differential expression of hap2 is associated with the development of B. bovis sexual forms. Overall these studies are consistent with a role of HAP2 in tick stages of the parasite and suggest that HAP2 is a potential candidate for a transmission blocking vaccine against bovine babesiosis.
Asunto(s)
Vectores Arácnidos/parasitología , Babesia bovis/genética , Babesia bovis/fisiología , Genes Protozoarios , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Rhipicephalus/parasitología , Animales , Babesia bovis/efectos de los fármacos , Babesia bovis/crecimiento & desarrollo , Bovinos/parasitología , Eritrocitos/parasitología , Femenino , Estadios del Ciclo de Vida , Factor 1 de Elongación Peptídica/genética , Regiones Promotoras Genéticas , Reproducción/efectos de los fármacos , Reproducción/genética , Xanturenatos/farmacologíaRESUMEN
BACKGROUND: Ticks are blood-feeding arthropods that can affect human and animal health both directly by blood-feeding and indirectly by transmitting pathogens. The cattle tick Rhipicephalus (Boophilus) microplus is one of the most economically important ectoparasites of bovines worldwide and it is responsible for the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Aquaporins (AQPs) are water channel proteins implicated in physiological mechanisms of osmoregulation. Members of the AQP family are critical for blood-feeding arthropods considering the extreme osmoregulatory changes that occur during their feeding. We investigated the pattern of expression of a newly identified AQP2 gene of R. microplus (RmAQP2) in different tick tissues and stages. We also examined in vivo the biological implications of silencing expression of RmAQP2 silencing during tick feeding on either uninfected or B. bovis-infected cattle. METHODS: In silico gene analyses were performed by multiple alignments of amino acid sequences and topology prediction. Levels of RmAQP2 transcripts in different tick tissues and stages were analyzed by reverse transcriptase quantitative PCR. Patterns of expression of RmAQP2 protein were investigated by immunoblots. Gene silencing was performed by RNA interference and in vivo functional analyses carried out by feeding ticks on either uninfected or B. bovis-infected cattle. RESULTS: RmAQP2 transcripts were found in unfed larvae, engorged nymphs, and salivary glands and guts of partially engorged females; however, of all tick tissues and stages examined, RmAQP2 protein was found only in salivary glands of partially engorged females. RmAQP2 silencing significantly reduced tick fitness and completely abrogated protein expression. The effect of RmAQP2 silencing on fitness was more pronounced in females fed on a B. bovis-infected calf than in ticks fed on an uninfected calf and none of their larval progeny survived. CONCLUSIONS: Collectively, considering the gene expression and tick fitness data, we conclude that RmAQP2 is critical for tick blood feeding and may be a suitable candidate target for the development of novel strategies to control R. microplus and tick-borne parasites.
Asunto(s)
Acuaporina 2/genética , Acuaporina 2/metabolismo , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Rhipicephalus/fisiología , Animales , Acuaporina 2/antagonistas & inhibidores , Proteínas de Artrópodos/antagonistas & inhibidores , Babesiosis , Bovinos , Enfermedades de los Bovinos , Conducta Alimentaria , Perfilación de la Expresión Génica , Silenciador del Gen , Datos de Secuencia Molecular , Rhipicephalus/genética , Análisis de Secuencia de ADNRESUMEN
Minimum postmortem interval estimations of a corpse using blow fly larvae in medicolegal investigations require correct identification and the application of appropriate developmental data of the identified fly species. Species identification of forensically relevant blow flies could be very difficult and time consuming when specimens are damaged or in the event of morphologically indistinguishable immature stages, which are most common at crime scenes. In response to this, an alternative, accurate determination of species may depend on sequencing and molecular techniques for identification. Chrysomyinae specimens (n = 158) belonging to three forensically important species [Chrysomya albiceps (Wiedemann), Chrysomya megacephala (F.), and Chrysomya marginalis (Wiedemann)] (Diptera: Calliphoridae) were collected from four locations in Egypt (Giza, Dayrout, Minya, and North Sinai) and sequenced across the mitochondrial cytochrome oxidase subunit I (COI) gene. Phylogenetic analyses using neighbor-joining, maximum likelihood and maximum parsimony methods resulted in the same topological structure and confirmed DNA based identification of all specimens. Interspecific divergence between pairs of species was 5.3% (C. marginalis-C. megacephala), 7% (C. albiceps-C. megacephala), and 8% (C. albiceps-C. marginalis). These divergences are sufficient to confirm the utility of cytochrome oxidase subunit I gene in the molecular identification of these flies in Egypt. Importantly, the maximum intraspecific divergence among individuals within a species was <1% and the least nucleotide divergence between species used for phylogenetic analysis was 3.6%. This study highlights the need for thorough and diverse sampling to capture all of the possible genetic diversity if DNA barcoding is to be used for molecular identification.
Asunto(s)
Dípteros/clasificación , Dípteros/genética , Patologia Forense , Variación Genética , Animales , Cadáver , Dípteros/crecimiento & desarrollo , Egipto , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Haplotipos , Humanos , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/clasificación , Larva/genética , Larva/crecimiento & desarrollo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Cambios Post Mortem , Análisis de Secuencia de ADNRESUMEN
The structure modulation of follicular cells and the ovarian changes during fourth larval instar and pupal stage of the malaria vector mosquitoes Anopheles pharoensis Theobald were investigated using the light and electron microscopy. The generative organs consist of a pair of polytrophic ovaries (OV), which are oblong, spindle-shaped bodies, lying dorsolaterally and occupying the region from the mid-fifth to the mid-sixth abdominal segment in the fourth larval instar, while in the pupal stage, each ovary (OV) is situated in the haemocoel of the sixth abdominal segment. It is an oblong body slightly larger in diameter; the lumen of the calyx becomes wider and central, and the pedicel (P) consists of one row of compact discoidal cells; meanwhile, in the fourth larval instar, the pedicel is without a lumen and consists of two rows of discoidal cells which are arranged as a short column between the follicle and calyx. The mean volume of the follicle in the fourth larval instar is 9.078 ± 3.0178 µm(3), meanwhile in the pupal stage being 12.051 ± 2.427 µm(3). The germarium (G) decreases in size in the pupal stage and contains a group of cells from which the oogonia differentiate, follicular cells which are similar to trophocytes, undifferentiated into one oocyte (O), which will develop into an egg and it is statistically the smallest one measured (0.058 ± 0.0041 µm(3), 0.303 ± 0.0086 µm(3)) in fourth larval instar and pupal stage, respectively as compared to the others within the follicle which will be accompanied as nurse cells (NC). The follicle is enclosed by a mononuclear flattened cells (follicular membrane), which have distinct boundaries. The vitellarium is differentiated into primary (F1) and secondary follicles (F2) in the pupal stage. The Golgi apparatus (GA) appears as discrete bits which are restricted to the perinuclear zone. The mitochondria (M) in the fourth larval instar are in the form of granules and short rods. They are perinuclearly distributed, forming a ring that surrounds the comparatively large nucleus. In the pupal stage, a similar condition to that described for the larva is observed, but with an increase in size and numbers, due to breaking up of rods into granules.
Asunto(s)
Anopheles/anatomía & histología , Folículo Ovárico/citología , Animales , Egipto , Femenino , Larva/anatomía & histología , Oocitos/citología , Ovario/citología , Pupa/anatomía & histologíaRESUMEN
The effect of larval and adult nutrition on survival and fecundity of the dengue vector Aedes albopictus Skuse (Diptera: Culicidae) were studied under laboratory conditions, the energy for the physiological activities for both male and female mosquitoes is provided largely by their reserves during larval stage and affected by adult diets. Two groups of larvae (A, B) were reared at 27 ± 3 degrees C, 70-80% R.H. and DL. 12: 12. Group (A) with 200 larvae (high larval diet) and Group (B) with 600 larvae (low larval diet). Ae. albopictus exhibited increased fecundity and egg hatch success. Immature development was quick. Immature survival was high, with lowest rate in the pupal stage. Highest longevity was observed in large females fed water + 10% sucrose solution (29.571 ± 0.415 days) while the lowest one was (1.3 ± 0.132 days) in starved small females. Large females have significantly (P < 0.001) higher fecundity than smaller females, regardless of whether the females were provided 10% sucrose solution or not (524 ± 0.203 eggs/group 159.714 ± 0.1997 eggs/group), respectively. The addition of 10% sucrose solution significantly (P < 0.001) increase the fecundity regardless of whether large or small females (657.9 ± 0.2198 eggs/group, 242.429 ± 0.119 eggs/group), respectively.
Asunto(s)
Aedes/fisiología , Virus del Dengue/fisiología , Aedes/virología , Animales , Tamaño Corporal , Femenino , Fertilidad , Larva/fisiología , Larva/virología , Longevidad , MasculinoRESUMEN
Since Culex pipiens quinquefasciatus is the main vector of lymphatic filariasis in tropics and subtropics, the identification and quantification of this mosquito is an important task. Scanning electron microscopy reveals that morphological changes during larval development as the number of comb scale varies greatly and their complexity increases from first to the fourth instar. Also, their structures are more complex with a varying number of subapical denticles. The amount of pecten shows modifications at different larval instars with regard to the number and complexity of their spines. The pecten teeth increase in their number and complexity during development. The number of lateral palatal brush filaments increases during larval development from the first to the fourth instar. The ventral brush of the abdominal segment X in the first and second instars is composed of two respectively three pairs of setae while the third and fourth instars have four pairs of sturdy setae.
Asunto(s)
Culex/ultraestructura , Vectores de Enfermedades , Estructuras Animales/ultraestructura , Animales , Culex/embriología , Larva/ultraestructura , Microscopía Electrónica de RastreoRESUMEN
During this study the surface morphology and morphometric of Culex pipiens pipiens (Lin.) and Culex quinquefasciatus (Say) eggs were described and compared using scanning electron microscopy for the first time. The results indicate that eggs of both species appear to be similar to great extent in surface morphology. However, morphometric provide 71.621% demarking attributes out of 44 attributes at various significant levels (P < 0.05-0.001), i.e., egg length, width and ratio of length/width, attributes of micropylar apparatus including corolla, disc, mound, tubercles size, and length of tubercular rows in micropylar region, size and density of tubercles, exochorionic pore in conical-shaped regions of eggs, and size of tubercular wheel units. Structurally, the additional presence of large tubercles strengthens the micropylar region to bear various collapsing forces in these species.
Asunto(s)
Culex/ultraestructura , Animales , Femenino , Microscopía Electrónica de Rastreo , Óvulo/ultraestructuraRESUMEN
The formation and uptake of the yolk protein in the oocyte of the Asian Tiger, Aedes albopictus mosquito was investigated. Light and electron microscopy of the ovaries at early resting stage as well as the structural changes associated with yolk formation were described 16 h after blood meal. The deposition of the yolk protein in the oocyte was correlated with a 15-fold increase in 138-µm pit-like depressions on the oocyte surface. These pits result by invagination of the oocyte cell membrane. They have a 20-µm bristle coat on their convex cytoplasmic side and a layer of protein on their concave extraoocyte space. The pits, by pinching off from the cell membrane become bristle coat vesicles which carry the adsorbed protein into the oocyte. These vesicles lose the coat and then fuse to form small crystalline yolk droplets, which subsequently coalesce to form the large protein yolk bodies of the mature oocyte. Preliminary radioautographs and certain morphological features of the fat body, ovary, and midgut, suggest that the midgut is the principal site of the yolk protein synthesis in A. albopictus.
Asunto(s)
Aedes/metabolismo , Proteínas del Huevo/metabolismo , Oocitos/metabolismo , Animales , FemeninoRESUMEN
The prevalence of Borrelia burgdorferi sensu lato (s.l.), the etiologic agent of Lyme borrelosis (LB), was determined for the first time in Egypt by using polymerase chain reaction (PCR). Questing 5243 hard and soft ticks were collected from animal farms throughout Giza Governorate. DNA from 500 individual tick species was extracted and PCR was performed. Primers verified from the sequence of German strain Pko of Borrelia afzelii were used. Fragments of 642 bp were generated and sequenced. The prevalence of B. burgdorferi sensu lato (s.l.) was 28% of examined soft and hard ticks. High infection rate (66%) of B. burgdorferi s.l. was observed in both nymph and adult soft ticks Ornithodoros savignyi. Beside, the role of hard ticks as potential vectors of Lyme disease in Egypt, where the infection rate was between 0.0-50.0%. Sequence analysis of PCR product of Borrelia burgdorferi sensu lato shares high degree of similarity in sequence compared to similar species in GenBank.
Asunto(s)
Grupo Borrelia Burgdorferi/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Garrapatas/microbiología , Animales , Secuencia de Bases , Egipto , Anotación de Secuencia MolecularRESUMEN
Christopher's stages of ovarian development are redefined in Culex pipiens quinquefasciatus to provide a more useful description of mosquito physiology. During this ovarian development, seven growing stages of the oocyte are observed in the adult stage. Two distinct periods of egg follicle growth, the previtellogenesis and vitellogenesis, are seen first. The gradual increase in the oocyte size during these phases is attributed to yolk deposition. During vitellogenesis, the oocyte and nurse cell nucleus are similar in size (stage I). Then, the oocyte enlarges and occupies one third of the egg follicle, while the nurse cell (NC) occupies the remaining two thirds. At this phase, the yolk granules (stage III) enlarge, occupying about half of the egg follicle, while the NC contain the other half (stage IV). An increase and accumulation of yolk granules leads to the filling of about three fourths of the egg bulk and makes the oocyte nucleus hardly visible during stage V. The oocyte undergoes a remarkable growth, occupying nine tenth of the length of the follicle in stage VI. Stage VII corresponds to a mature cigar-shaped egg and contains two kinds of yolk globules: a large one and a small one. The oocyte and the NC at all stages are surrounded by a single layer of epithelial cells which increase in number and form the vitelline membrane and the chorion. The mitochondria in the NC, oocyte, and follicular epithelium appear in light microscopy as granules of different sizes. The Golgi apparatus appears in different sizes and shapes, since in the early stages of development, it is restricted to the perinuclear zone. When the development proceeds, it enlarges in size, spreads over the whole cytoplasm, and participates in the formation of the yolk.
Asunto(s)
Culex/citología , Culex/ultraestructura , Oocitos/citología , Oocitos/ultraestructura , Ovario/citología , Ovario/ultraestructura , Animales , Femenino , Microscopía , Microscopía Electrónica de Transmisión , Orgánulos/ultraestructuraRESUMEN
The bean skink lizard Mabuya quinquetaeniata quinquetaeniata (Family: Scincidae) collected from Abu-Rawash, Giza was infected with the protozoon Hepatozoon gracilis. The mean percent of infection rate all over the year was 12.57% with the highest peak was in October (33%), meanwhile the lowest infection rate was detected in June (2.1%) and no infection was detected in February. Microscopical examination of experimentally infected Culex (C.) pipiens L smears revealed the presence of gamogony and sporogony stages of H. gracilis in their haemoceal. Cx. pipiens was capable to transmit H. gracilis to non-infected M. q. quinquetaeniata under laboratory conditions (21 +/- 1 & 60-70% R.H) after a prepatent period of 32 +/- 1 days from the infective bite. Various developmental schizogony stages were detected both in the lung endothelial cells and liver parenchymal cells. Developmental stages of H. gracilis in both vector or vertebrate host were described. Cx. pipiens was unable to transmit H. gracilis to snake, Psammophis schokari present in the same area of the bean skink lizard.
Asunto(s)
Coccidios/crecimiento & desarrollo , Coccidiosis/transmisión , Culex/parasitología , Insectos Vectores/parasitología , Lagartos/parasitología , Animales , Mordeduras y Picaduras , Egipto , Humanos , Estadios del Ciclo de Vida , Estaciones del AñoRESUMEN
Laboratory observations on the effect of Hepatozoon gracilis on the egg production of the mosquito Cx. (Cx.) pipiens Linneaus under laboratory conditions revealed that H. gracilis infected mosquitoes produced significantly fewer eggs than uninfected ones. The egg production decreased as parasite burdens increased. Reduction in blood meal size in infected females did not reduce fecundity. No size differences was detected between oocyst-infected and uninfected females although sporozoite positive females were significantly large. Preoviposition period was affected significantly, while incubation period and percentage of egg hatching showed no significant changes. The longevity of female infected mosquitoes decreased insignificantly than in uninfected ones.