Aphis nerii, though native to the Mediterranean region, is an invasive pest species throughout much of the world. Feeding primarily on Oleander (Nerium oleander) and Milkweed (Asclepias spp.) under natural conditions, these plants are unlikely to support aphid populations year round in the southern US. The objective of this study was to describe the genetic variation within and among US populations of A. nerii, during extinction/recolonization events, to better understand the population ecology of this invasive species.The importance of genetic diversity in successful biological invasions is unclear. In animals, but not necessarily plants, increased genetic diversity is generally associated with successful colonization and establishment of novel habitats. The Oleander aphid, A. nerii “superclones”. Genotypic variation was absent in all populations  or “clone”) and the genetic composition of only one population completely changed across years. There was no evidence of sexual reproduction or host races on different plant species.We used five microsatellite markers to assess genetic diversity over a two year period within and among three aphid populations separated by small (100 km) and large  geographic distances on two host plant species. Here we provide evidence for Aphis nerii is a well established invasive species despite having extremely low genetic diversity. As this aphid appears to be obligatorily asexual, it may share more similarities with clonally reproducing invasive plants, than with other animals. Patterns of temporal and geographic genetic variation, viewed in the context of its population dynamics, have important implications for the management of invasive pests and the evolutionary biology of asexual species. While studies are few, genetic diversity is believed to facilitate successful biological invasions Aphis nerii Boyer de Fonscolombe, is a pest of several plant families including Apocynaceae (Nerium and Vinca), Asclepiadaceae , Asteraceae, Convolvulaceae, Euphorbiaceae, and Rutaceae Aphis nerii has since become a common invasive species in warm temperate and tropical regions of the world Aphids are valuable study systems for investigating the roles of genetic variation and phenotypic plasticity on population, ecological, and evolutionary dynamics Nerium oleander (Apocynaceae), is a common ornamental plant in southern and coastal states and frequently grows along US highways A. nerii sequesters for defense, and are unable to support aphid populations throughout the year In the United States, the Oleander aphid commonly infests two plant families, Apocynaceae and Asclepiadaceae A. nerii inhabiting species of milkweed that differed in the amount cardiac glycosides that they possess A. nerii among different host plant families such as Milkweed and Oleander, to our knowledge, have not been examined.Aphid fitness tradeoffs among host plant species results in selection for host fidelity which can inhibit gene flow and result in the development of host races; i.e., host-associated population genetic structure Aphis nerii is believed to be an obligate parthenogen; males have never been found in natural populations Aphid species vary in their mode of reproduction from obligate to cyclical parthenogens, but less than 3% of species are strictly clonal A. nerii, produce winged forms in response to overcrowding and/or decreasing host plant quality Aphis nerii, however, is found in patchily distributed habitats throughout the United States, and both Oleander and Milkweed are unable to support aphid populations year round except in the southernmost latitudes of the US The genetic structure of aphid populations is shaped spatially and temporally by habitat distribution, dispersal capabilities, and life-cycle. Many aphids, including A. nerii with the goal of gaining insight into the population dynamics, life history, and ecology of this well-established invasive species. In so doing, we asked the following questions: 1) Is there any evidence of sexual reproduction in US populations of A. nerii?; 2) Do aphids inhabiting different hosts comprise host races?; 3) Does the patchy distribution of suitable habitat result in population genetic subdivision over small and/or large geographic ranges?; and 4) Is there temporal variation in population genetic structure? To address these questions, we used microsatellite markers to assess genetic diversity over a two year period within and among three populations separated by roughly 100 km and 3,700 km on two host plant species.The aim of this study was to characterize genetic variation within and among southern US populations of A. nerii. Whilst genetic variation was extremely low within and among populations, the genetic composition of one population was found to change drastically over time. Our findings suggest that A. nerii is an efficient colonizer that demonstrates true metapopulation dynamics.Here, we report no evidence of sexual reproduction or the existence of host races in N. oleander) at two locations in Georgia and one location in central California, June-August 2008, 2009. This “overwintering” period between samples allowed us to assess temporal genetic variation, and determine if sexual reproduction occurred. Sampling locations were: Statesboro, Georgia ; Tybee Island, Georgia ; and Concord, California . Sampling areas consisted of multiple patches of Oleander plants in close proximity but intermittently up to 2.5 km apart. To survey genetic diversity within each population, aphids were collected from different parts of the same plant and from as many different plants as possible. To assess genetic variation among aphids inhabiting different host plant species, aphids were collected from Milkweed (Asclepias amplexicaulis) at a second site in Statesboro, Georgia  in June 2008.Aphids were collected from Oleander  using primers originally designed for the Cotton/Melon aphid, 2. Reactions consisted of a 4 min denaturation at 94°C, followed by 35 cycles of 30 s at 94°C, 35 s at 58°C, and 45 s at 72°C, and a final 10 min extension step at 72°C.DNA was extracted by macerating individual aphids in 70 µl cell-lysis/proteinase-K buffer  followed by incubation for 1 h at 65°C and 15 min at 99°C (Lee and Frost 2002). PCR reactions were carried out in 20 µl final volumes with 2.5 mM MgClPCR products were run on small (15 cm×17 cm×0.8 mm), non-denaturing TAE  buffered polyacrylamide gels of either 9% or 10% concentration, with the lower portion of the gel supplemented with EnhanceIT polymer . Gels were run for 2 h at 30 mA, stained with ethidium bromide, and visualized on a UV light box. Allele sizes at each locus were estimated using the M3 size standard .FISFST and RST using the program FSTAT v. 2.9.3 F-statistics was tested using the randomization procedure available in FSTAT using 5000 permutations. Estimates of genetic distance between multilocus genotypes  (δμ2 and DSW) were calculated according to Shriver et al. O) to that expected under conditions of sexual reproduction (GE), as described by Stoddart and Taylor Observed and expected heterozygosities were calculated for each locus in each population. Deviations from Hardy-Weinberg expectations were tested according to Guo and Thompson A. nerii. The number of alleles was low; 3 to 4 alleles per locus. Three of the five loci each had an observed heterozygosity (HO) of 0.143, the two loci an HO of 1. Null alleles were not detected for any loci in any populations sampled.All five microsatellite loci used in this study were polymorphic in Aphis nerii. Only 2 multi-locus genotypes (MLGs) or “clones” were detected among the 319 individuals assayed across all populations and both years  suggesting that the MLGs do not share a close genealogical relationship Despite the low level of genotypic diversity observed, the genotypes of the two MLGs were divergent. That is, the two MLGs did not share any alleles, clearly indicating that they were not the product of recombination through sexual reproduction. Estimates of genetic distance, based on the stepwise mutation model, were large (δμO/GE) from 0.141 to 0.048 . Fourth, estimates of FIS were −1.0 for all populations.No evidence of sexual or mitotic recombination was found. First, no homozygous allelic arrangements were found at any of the heterozygous loci . Second,A. nerii from two common host plants (Oleander and Milkweed) from Statesboro, GA . All samples from both hosts consisted of MLG 1, indicating that there is no host associated subdivision at this location   .FST = 0, RST = 0)   . The Tyb= 0.787) . During A. nerii to better understand the ecology, life history, and population dynamics of this well established invasive species. We found that Oleander aphids are remarkably invasive throughout the southern United States, with extremely low genetic diversity.Small, genetically-uniform populations are subject to ecological and evolutionary forces  which threaten population persistence, in both native and introduced habitats Aphis nerii is believed to be obligately parthenogenetic, based on the complete absence of males under natural conditions A. nerii in the southern US.A. nerii. For example, long term asexual populations of the pea aphid, Acyrthosiphon pisum (Harris) in Japan were composed of five to seven MLGs, and cyclically parthenogenetic populations harbored much greater diversity than did asexual populations A. pisum, both within and among populations The most striking pattern observed in this study was the low level of genotypic diversity within and among populations over a large geographic area. We observed only two MLGs and a maximum estimate of genotypic diversity (G/N) within any population of 0.05. Several similar studies of other aphid species using comparable numbers of loci (4 to 7) have revealed higher levels of genetic variation than that observed in et al.A. pisum in Chile. Among the 432 individuals sampled over a 570 km range, 16 MLG's were identified with three MLG's comprising≈90%  of the diversity. Compared to previous studies, the pattern we observe in A. nerii is extreme even for “superclones”. Within any sampling year, each population consisted of a single MLG. In 2008, all populations sampled from both California and Georgia showed only a single MLG (MLG 1). In 2009, populations from Statesboro, Georgia and California comprised the same MLG (MLG 1), whilst a population from Tybee Island, Georgia comprised a single but entirely different MLG (MLG 2).Aphid populations are sometimes composed of a small number of dominant genotypes (“clones”) and many low frequency (rare) genotypes A. nerii has been assessed within and among populations from California, Iowa, and Puerto Rico Variation of morphometric and life history traits in A. nerii likely results from successive genetic bottlenecks or founder events. Subsequent clonal propagation and rapid spread of genotypes throughout the host range would be followed by rapid clonal competition/selection resulting in a few geographically-widespread and dominant clones  is the most common host of A. nerii in the northern US where N. oleander grows infrequently Asclepias species are perennial plants that sprout in Spring, bloom in early Summer, and then set seed and die-back in the Fall. The life history of this host plant would require A.nerii to have a secondary host (though this has not previously been reported) or to produce sexuals to produce overwintering eggs . On both Oleander and Milkweed, a regular pattern of colonization, rapid increase, and population extinction would be expected.Milkweed  Aphis gossypii Glover A. nerii populations, we would have expected to see genetic variation between populations inhabiting Oleander and those inhabiting Milkweed, regardless of geographic proximity. Our findings suggest that there is no selection for host specificity and that A. nerii is a polyphagous, i.e. generalist species, although more data are required to confirm this contention. This pattern is consistent with Lynch Groeters A. nerii surveyed in this study show the genetic signature of obligate parthenogenetic reproduction, supporting previous reports that the species is indeed obligately asexual. Within any sampling period, each population was composed of a single MLG. This level of variation is remarkably low compared to the variation observed in other aphid species presumed to be largely or completely asexual A. nerii is a generalist species with strong dispersal capabilities. As patches of host plants are colonized by few individuals that reproduce rapidly through parthenogenesis, founder individuals likely come from a source population(s) characterized by low genetic diversity.In summary, populations of A. nerii provides great insight for the management of invasive pests and the population dynamics of clonal organisms.Despite having extremely low genetic diversity Aphis nerii is a well established invasive species. Understanding the temporal and geographic genetic variation of


Eutric Cambisol developed from loess (South-East Poland). Two doses of both dairy sewage sludge (20 Mg·ha−1 and 26 Mg·ha−1) and of mineral fertilizers containing the same amount of nutrients were applied. The same soil without any amendment was used as a control. The soil under undisturbed native vegetation was also included in the study as a representative background sample. The functional diversity  was assessed using such indices as Average Well Color Development (AWCD), Richness (R) and Shannon–Weaver index (H). These indices were calculated, following the community level physiological profiling (CLPP) using Biolog Eco Plates. Soil dehydrogenase and respiratory activity were also evaluated. The indices were sensitive enough to reveal changes in community level physiological profiles due to treatment effects. It was shown that dairy sewage amended soil was characterized by greater AWCD, R, H and dehydrogenase and respiratory activity as compared to control or mineral fertilized soil. Analysis of variance (ANOVA) and principal component analysis (PCA) were used to depict the differences of the soil bacterial functional diversity between the treatments.The aim of the present work was to assess the influence of organic amendment applications compared to mineral fertilization on soil microbial activity and functional diversity. The field experiment was set up on a soil classified as an  That is also reflected in the upswing of the number of sewage treatment plants that produce continually increasing amounts of sewage sludge. This vast volume of sewage sludge must be neutralized and utilized in an appropriate manner ,2. SewagLand application of sewage sludge is one of the most important disposal alternatives, but potential availability of heavy metals, toxic compounds and pathogenic bacteria often restricts its uses –6. Some Monitoring the soil microbial community and activity can be a powerful tool for understanding basic and applied ecological contexts. Rapid growth of microorganisms in the soil allows for comprehensive study of community interactions much more readily than in a plant or animal system , and, eset al. [et al. [Nevertheless, it seems necessary to take under consideration the rapid community-level cultural approach, subsequently called CLPP, developed by Garland and Millis ; for 95 et al.  and Lehm [et al.  indicate [et al.  proposed [et al. ,20. The  [et al. . On the  [et al. ,21 noticIt is assumed, that agricultural management practices must be compatible with the principle of sustainable development. They must also promote long-term fertility and productivity providing nutrient supplements, fixed and mineralized by the microbiota ,23. OrgaThe aim of the present work was to assess the response of soil microbial activity and functional diversity to dairy sewage sludge application and mineral fertilization. The study included determination of dehydrogenase and respiratory activity and CLPP using the Biolog EcoPlates™ system. This research offers important data that supports the application of sewage sludge to agricultural land.2.2.1.Eutric Cambisol soil type developed from loess. The experiment sites were situated in the vicinity of a dairy sewage treatment plant in Krasnystaw, South-East Poland . The experiment was established in an agricultural field that had been in conventional tillage agriculture for at least past 50 years with crop rotations including selected cereals, root crops and papilionaceous crops. The research area has rather uniform soils with respect to genesis and textural composition [The field experiment was conducted on an Undisturbed native vegetation established on the experimental plots abandoned from agricultural use in 1996 (UND),Soil with neither, mineral fertilization or dairy sewage sludge, control (C),−1, 80 kg·P·ha−1, 120 kg·K·ha−1 ,Mineral fertilization amendment 130 kg·N·ha−1, 100 kg·P·ha−1, 160 kg·K·ha−1 ,Mineral fertilization amendment 170 kg·N·ha−1 plus 80 kg·K·ha−1 in order to complement the amount of nutrients to the same level as in MFSD ,Dairy sewage sludge amendment 20 Mg·ha−1 plus 110 kg·K·ha−1 in order to complement the amount of nutrients to the same level as in MFLD (termed DSSLD–dairy sewage sludge large dose).Dairy sewage sludge, 26 Mg·haThe treatments were as follows:The main characteristics of the dairy sewage sludge (DSS) are given in 2.2.org.) was determined using the Tiurin method [Soil total organic carbon  values from each plate well [Microbial response in each microplate that expressed average well-color development (AWCD) was determined as follows :AWCD=∑Oate well ,36.i.e., the richness and evenness of response) were calculated using an OD of 0.25 as threshold for positive response [i is the ratio of the activity on each substrate (ODi) to the sum of activities on all substrates ∑ODi. Plate readings at 48 h of incubation were used to calculate AWCD, R and H, since it was the shortest incubation time that allowed the best resolution among the treatments.Richness (R) values were calculated as the number of oxidized C substrates, and the Shannon–Weaver index values (H)  carbohydrates; (2) carboxylic and acetic acids; (3) amino acids; (4) polymers; and (5) amines and amides according to Weber and Legge  Table 3Table 3. et al. , were inet al. . For eacet al. ,39.2.5.et al. [Average well-color development, richness and Shannon–Weaver indices, dehydrogenase and respiratory activity were analyzed by ANOVA and mean comparisons between treatments were performed using Tukey’s mean separation test at P < 0.05. Principal component analysis (PCA) to analyze CLPPs was performed on normalized and transformed absorbance data for each well, according to Weber et al. . Transfo3.As demonstrated by et al. [Baudoin et al.  indicateIn order to determine the extent of differentiation of treatments from the control and the undisturbed sites with regard to carbon source catabolism, each of the amendment application rates was subjected to principal component analysis. The first and the second principal component (PC1 and PC2) explained 36.7% and 26.5% of data variance, respectively, with a small dose of the mineral and organic amendments. At the large dose of the amendments the corresponding values were 27.6% and 20.3% for PC1 and PC2 . The ploThe substrates situated on microtiter plates were divided into five main groups: carbohydrates, carboxylic and acetic acids, amino acids, polymers, amines and amides. Their relative contributions in soil amended with dairy sewage sludge and mineral fertilization were examined. Comparing the guild utilizations for particular treatments the group shifts are similar for the three treatments . These results suggest that bacterial communities in all treatments are similar. The shifts of dairy sewage sludge amended and undisturbed soil are different from the control and mineral fertilized treatments shifts, which clearly confirms a microbial response to the perturbation of treatments. The data suggest that composition of microbial communities shows susceptibility to change under studied treatments. Dairy sewage sludge application to the soil seems to boost its microbial communities and increase its predisposition to recover it towards UND communities. We found a strong contribution of carbohydrates and amino acids throughout the DSS amended and undisturbed soils. The average absorption value of carbohydrates declined within increase in the average absorption value of amino acids in MFSD and amines and amides in control soil. It suggests that carbohydrates and amino acids were incorporated into soil with dairy sewage sludge. The results indicated that the carbohydrates (Carb) and amines and amides (A&A) groups were the most intensively metabolized, especially in the sewage sludge-treated soil and control soil, respectively. Generally, the soil under undisturbed native vegetation showed similar utilization level of all the groups of substrates. The results also indicated that dairy sewage sludge amendment appreciably increased the level of carbohydrates and decreased the level of amines and amides usage compared to control soil. There was no significant effect of the amendments dose on substrate categorized utilization pattern.et al. [Low carbon availability is frequently a limiting factor for microbial growth ,41,42. Iet al. ), were set al. . As can Community level physiological profiles showed that potential metabolic diversity was significantly enhanced in the soil treated with dairy sewage sludge compared to chemical fertilizer as shown by values of AWCD and R index. Microbial activity, expressed as AWCD, was higher in dairy sewage sludge treated soil and undisturbed treatment compared to the control and mineral fertilized soils. Taking into consideration that the amount of nutrients introduced with the dairy sewage sludge and mineral fertilizers was the same, the results indicate that the increased functional diversity may be due to an additional supply of carbon to soil through the sludge on the DSS plots.d-malic acid and phenylethylamine) for small dose of amendments and two carbon sources  for large dose of amendments from the 30 studied. These results indicate that carbon sources like carboxylic acids and amines had influence on community level physiological profiles, suggesting grater carbon availability from these sources.CLPP analysis using principal component analysis showed that the DSSLD and the undisturbed soils were different than the control and MFLD soils by PC1 , explaining 36% of the variance. This implies differences in microbial communities between the two groups of treatments that can result from a greater availability of C in the former. Also, PCA performed on data from each of the treatments with small dose applications showed that plots amended with dairy sewage sludge and soil under undisturbed native vegetation grouped together and differentiated from the control and mineral fertilized soil. This suggests that greater carbon availability due to dairy sewage sludge incorporation could explain the increase in microbial functional potential. Second principal component analysis was correlated with three carbon sources  of the soil microbial population and at the same time can be a useful opportunity for its recycling. Applicability of the sludge used in this study can be enhanced by its low concentrations of heavy metals.The close correlation between catabolic potential of microorganisms and functional microbial diversity was observed in soil amended with dairy sewage sludge and mineral fertilization.Continuation of the experiments will allow determining how the dairy sewage sludge may affect microbial community and functional potential in a longer time span.The results of this study showed the following:


This is a case of a 71-year-old woman, who presented with a large abdominal mass. Microscopic examination revealed metastatic renal cell carcinoma in an otherwise massively enlarged benign ovarian tumour of mixed mucinous cystadenoma and Brenner tumour. Clinicopathological and radiological assessment together with a thorough evaluation of gross and histological features, including the use of immunohistochemical stains, is essential to confirm the diagnosis and rule out the possibility of primary clear cell carcinoma of ovary, as this has important prognostic and therapeutic implications. Renal cell carcinoma (RCC) is the most common malignant tumour of the kidney. It constitutes 75% of renal neoplasms in adults and 3% of all adult malignancies . Approxi2.A 71-year-old woman presented clinically with abdominal distention and weight loss. On physical examination, the patient had a large abdominal mass. Blood biochemistry showed Urea 6.5 mmol/L, Creatinine 77 umol/L, Sodium: 136 mmol/L, Potassium 5.2 mmol/L, and GFR 79 mL/min/1.7 mμg/L . The abdominal/pelvic ultrasound revealed a right ovarian mass which was confirmed on CT scan.The tumour markers showed CA125 was 7.4 KU/L , CA19.9 = 19 KU/L , and CEA = 1.2 Laparotomy was performed, and the ovarian mass together with right fallopian tube were excised. The mass weighed 9.5 kg and measured 290 × 115 × 120 mm. Sectioning of the mass showed a multilocular cystic areas containing amber coloured, thick mucoid material alternating with multiple, solid firm nodules within the fibrous stroma of the walls. Histological examination showed a multilocular cystic ovarian tumour which consisted of mixed benign mucinous cystadenoma and Brenner tumour Figures  and 2. TThe grading and nature of this tumor was difficult to assess on conventional H&E sections. The differential diagnoses were clear cell carcinoma of the ovary, carcinoid, and Sertoli cell tumor.The clear cells were positive for immunostains CD10, Pan CK, Cam5.2, Vimentin, and EMA. The cells were negative for CK7, CK20, GP200, ER/PR, and S100. The histology and the immunostaining profile were suggestive of a metastatic renal cell carcinoma—clear cell type which is present in a benign mixed mucinous cystadenoma and Brenner tumour Figures . Further investigation of the patient revealed the presence of a tumour in the right kidney which was confirmed as renal cell carcinoma.Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. It constitutes 85% of renal neoplasms in adults and 3% of all adult malignancies . It usuaSometimes, mucinous cystadenomas are associated with other types of primary ovarian tumours such as Brenner tumour. The latter constitutes about only 2% of all ovarian tumours and has been reported in 20% of cases in association with other tumours such as benign serous, mucinous cystadenoma, or benign cystic teratoma , 8. Secondary tumours of the ovary constitute only around 6% of ovarian tumors. The usual cancers that metastasize to the ovaries are those arising from stomach, colon, breast, and lymphomas . Renal cell carcinomas rarely metastasize to the ovaries because these tumors usually occur during the 6th and 7th decades of life, where sclerosis of the ovarian vessels had usually occurred. Therefore, due to its rarity, clear cell carcinoma of the kidney when metastasizes to the ovaries is usually misdiagnosed as primary clear cell carcinoma of the ovary . Sometimes, it can be difficult to differentiate between renal cell carcinoma—clear cell type and clear cell carcinoma of the ovary. However, the presence of certain histological criteria can help pathologists to differentiate between the two tumors. Tubulocystic pattern with the presence of “Hobnail cells” and extracellular mucin are more in favour of clear cell carcinoma of the ovary, whereas the presence of solid sheets of clear cells and thin wall blood vessels surrounded by a pseudocapsule formed by the compressed parenchyma and fibrous tissue are all in favour of renal cell carcinoma—clear cell type . The differential diagnosis of primary clear cell tumour of the ovary includes primary clear cell carcinoma, Sertoli cell tumour, dysgerminoma, Lipoid cell tumour, Hilus cell and Leydig cell tumours . DespiteOur case highlights an unusual presentation of renal cell carcinoma that has metastasized to the ovary which was extensively involved by a mucinous cystadenoma associated with Brenner tumour.In conclusion, a thorough clinical history, with careful evaluation of gross and microscopic features of the clear cell ovarian tumour, and use of immunohistochemical stains are extremely important in the diagnosis of rare metastatic renal cell carcinoma to the ovary. It is also important to consider the possibility of renal cell carcinoma in the differential diagnosis of any suspicious abdominal mass, in view of the unpredictable biological behaviour of this tumour .


Postoperative cognitive impairment is a common complication after cardiac and major non-cardiac surgery in the elderly, but its causes and mechanisms remain unclear. The purpose of the current study was to use resting-state functional magnetic resonance imaging (fMRI) to explore changes in the functional connectivity, i.e. the synchronization of low frequency fluctuation (LFF), in an animal model of cognitive impairment in aged rats.Aged (22 months) rats were anaesthetized with 40 µg/kg fentanyl and 500 µg/kg droperidol  for splenectomy. Cognitive function was assessed using Y maze prior to operation and on postoperative days 1, 3 and 9. To evaluate functional connectivity, resting-state fMRI data were acquired using a 3T MR imaging system with a 4 channel phase array rat head coil.Cognitive function was impaired at postoperative days 1 and 3 compared with preoperative. Significant synchronized LFF was detected bilaterally in the primary somatosensory cortex and hippocampus preoperatively. By contrast, no significant LFF synchronization was detected in the right primary somatosensory cortex and right hippocampus on postoperative days 1 and 3, although the pattern of functional connectivity had become almost normal by day 9.Splenectomy performed under neuroleptic anaesthesia triggers a cognitive decline that is associated with altered spontaneous neuronal activity in the cortex and hippocampus. Postoperative cognitive dysfunction (POCD) is the impairment of perception, memory, and information processing after surgery Functional magnetic resonance imaging (fMRI) offers a useful perspective on brain function in vivo with high spatial resolution, and this is the approach we take here. Recently, spontaneous LFF in resting-state fMRI signals has been studied in the preclinical phase of neurodegenerative disease Postoperative cognitive decline has mainly been studied using either simple neuropsychological and behavioral methods −1 and oxygen saturation at 95–100% . After recovery, animals were returned to their cages and housed individually. Surgical procedures were approved by the Institutional Animal Use and Care Committee of Chinese Academia of China at Chengdu.Female aged Sprague-Dawley rats  were maintained in the temperature-, light/dark-controlled Animal Facilities of Chinese Academy of Science at Chengdu, China, with free access to rodent chow and water. Animal care was approved in accordance with the guidelines for Care and Use of Laboratory Animals in China.Splenectomy is chosen as a standardized organ removal intervention. Rats were anaesthetized with 40 µg/kg fentanyl and 500 µg/kg droperidol . A small incision was made in the left upper abdominal quadrant, the spleen was isolated, the arteries and veins ligated, the spleen removed and the wound closed by suture. Throughout surgery the animals were positioned over a heated pad: core temperature was monitored and maintained at 37–38°C, respiration rate at 80–100 breaths minCognitive function was assessed as spatial memory using a Y maze prior to operation and on postoperative days 1, 3 and 9 according to the method developed and described in detail by Wan 2-weighted images were acquired using a 3D turbo spin echo sequence . Functional images were acquired using single shot spin echo EPI sequence . The paradigm consisted of 5 dummy scans to reach steady state followed by 120 scans during rest, for a total experiment time of 225 seconds.fMRI was carried out prior to operation and on postoperative days 1, 3 and 9. For imaging, animals were anaesthetized with intraperitoneal chloral hydrate 300 mg/kg and placed in the scanner with an MR-compatible fixation device securing the head with a tooth bar. Body temperature was maintained at 37–38°C using warm air. fMRI data were acquired using a 3.0T MR imaging system  with a 4 channel phase array rat head coil. TpCO2 and pO2 using a blood gas analyzer .To monitor physiological responses, the femoral artery was cannulated (n = 6), and samples of arterial blood taken at the beginning and end of the scanning for measurement of pH, www.fil.ion.ucl.ac.uk/spm/software/) including slice timing, head-motion correction, spatial normalization to a standard rat brain template marsbar.sourceforge.net/), including low-pass filtering, seed region identification and generation of correlation  maps. Resting state time courses were first low-pass filtered with 0.08 Hz cutoff P<2.5×10−5 with reference waveform, taking into account the reduced degrees of freedom in the low-pass filtered data) to detect areas of LFF higher than the global mean.Image data was preprocessed using statistical parameter mapping , respectively, decreasing to a number not significantly different from preoperative on day 9  or in pO2 (169±4 vs 168±2 mmHg) .There were no significant changes between the start and end of the scan in pH (7.4±0.02 vs 7.4±0.02), in P<0.001, uncorrected) was detected bilaterally in the primary somatosensory cortex (SI) and hippocampus. By contrast, no significant LFF synchronization was detected in the right primary somatosensory cortex and right hippocampus on postoperative days 1 and 3 , but recovers by day 9.Excluding data from one rat which showed large nonlinear baseline drift, the results for 17 rats are shown in  1 and 3 , althoug 1 and 3 . This paTo demonstrate the synchronization of LFF in bilateral SIs preoperatively and on postoperative day 9, in vivo.Postoperative cognitive decline, a distressing complication after cardiac surgery or non-cardiac surgery, is independently associated with poor short- and long-term outcomes Evidence from both human and animal studies that some regions exhibiting task-related deactivation are functionally connected in the resting state viz. spatiotemporal correlations between spatially remote neurophysiological events. Although the origin of synchronized LFF of BOLD has not been fully elucidated, some investigators attribute it to spontaneous neuronal activity Synchronized LFF of BOLD fMRI signals are believed to reflect functional connectivity, In our study, to demonstrate the synchronized LFF in the BOLD signal in bilateral Sis preoperatively and on postoperative day 9, the timecourse of the seed ROI in the SI in one of the hemisphere is compared with the average timecourse of the SI ROI in the opposite hemisphere. As indicated in During the scan we used chloral hydrate (300 mg/kg), administered intraperitoneally (requiring no catheterization) to produce temporary sedation and muscle relaxation In our studies, the number of trials in the Y maze test was significantly increased on postoperative days 1 and 3, consistent with the changes in functional connectivity in the cortex and Hp. This is interesting given that the earliest functional manifestation of neuronal damage in the brain is a decline in the hippocampal and cortical functions of information storage and recall and cognitive processing A limitation of this study is that it did not include a group that underwent anesthesia without surgery, or with sham surgery. Clearly further studies are needed to establish the causation and clinical relevance of our observation, including other anaesthetic regimens, and other types of surgery.In conclusion, in this rat model of cognitive impairment, anaesthesia and surgery triggers a transient neurocognitive decline , at the same time as reduced synchronization of temporal fMRI correlations in the SI and Hp. Splenectomy performed during neuroleptic anaesthesia triggers a cognitive decline that is associated with altered spontaneous neuronal activity in the cortex and hippocampus. The ability to detect noninvasively abnormalities of functional connectivity offers a way of exploring the mechanisms of postoperative cognitive dysfunction in patients.


Microsatellite instability (MSI) has been found in a range of human tumors, and little is known of the links between MSI and herpesvirus. In order to investigate the relationship between MSI and Gallid herpesvirus 2 (GaHV-2)-induced lymphoma, fifteen Marek’s disease (MD) lymphomas were analyzed through using 46 microsatellite markers, which were amplified by PCR from DNA specimens of lymphoma and normal muscular tissues from the same chicken. PCR products were evaluated by denaturing polyacrylamide gel electrophoresis for MSI analysis. MSI was proved in all lymphomas, at least in one locus. Thirty of the 46 microsatellite markers had microsatellite alterations. These results suggested that GaHV-2-induced lymphoma in chickens is related to MSI, and this is the first report to demonstrate that MSI is associated with the GaHV-2 induced lymphoma in chicken. Epstein-Barr virus (EBV) is the etiological agent of nasopharyngeal carcinoma (NPC), African Burkitt’s lymphoma, posttransplant lymphomas (PTLD), Hodgkin’s disease, and some gastric cancers Microsatellite instability (MSI) is defined as a change of any length caused by either the insertion or deletion of repeating units, in a microsatellite within a tumor when compared to normal tissue The information available regarding the relationships between herpesvirus and MSI in carcinogenesis is controversial. Chang et al found that EBV-positive cases in gastric carcinomas showed no MSI positivity Specific pathogen free (SPF) White Leghorn Chickens  were kept at the animal isolation facility at Nannong Hi-Tech Co., Ltd . A virulent strain of MDV (GaHV-2) J-1 at passage 32, kindly provided by Merial Animal Health Co., Ltd , was used to infect the chickens.2. The experiment was terminated 58 days after infection. All animal experiments were carried out in dedicated negative pressure rooms and conducted strictly in accordance with the laboratory animal guidelines. The protocol was approved by Laboratory Animal Management Committee of Jiangsu Province.After 21 days post-hatch, forty SPF White Leghorn chickens were intraabdominally inoculated with 1,000 plaque-forming units of MDV J-1 and housed in the isolation facility in separate rooms. All birds were evaluated daily for symptoms of MD, and were examined for gross MD lesions. Chickens which present ataxia or paralysis and moribund state were euthanized by COTissues samples were removed and collected from chicken euthanized during the experiment period and termination, including the liver, spleen, kidney, heart, lung, peripheral nerves, skin, gonads, thymus and bursa of fabricius. Different tissue samples were fixed in 10% formalin and embedded in paraffin wax via a routine process. All sections were stained with hematoxylin and eosin (H&E) and histopathologically examined through using an optical microscope.Microsatellite instability was detected through using gel-based nonradioactive methods described by Shang et al. Tumor specimens that were verified visually and histopathologically were collected for MSI analysis. The genomic DNA of tumors and normal muscular tissue samples from the same chicken were extracted with TIANamp Genomic DNA kit , according to the manufacturer’s instructions. Their concentrations were determined by using a BioPhotometer plus .Forty-six microsatellite markers , which s3 according to the method of Sanguinetti et al PCR products were denatured at 95°C for 5 mins with gel loading dye, and put immediately on ice for 5 mins prior to loading. About 3 µl of the PCR products were loaded onto a 12% denaturing polyacrylamide gel. The gels were run on the DCode universal mutation detection system  in 1×TBE at 45°C at 180V. After electrophoresis, the gels were stained with AgNOClinical signs of infection by GaHV-2 appeared among the treated chickens, including loss of appetite, depression, pallor, and paralysis of the wings, legs and necks. Death occurred in the second week after post-infection, and reached a peak during the 4th and 5th week. Morbidity was 75%, and mortality was 73.3% when the experiment was terminated. Pathological examination of the chickens tissues revealed that fifteen chickens  had significantly widespread, diffuse lymphomas involving the liver (n = 9), gonads (n = 12), spleen (n = 9), kidneys (n = 15), proventriculus (n = 6) and heart (n = 9). Tumors were found in twelve chickens after death or euthanasia and in three chickens after euthanasia at the predetermined time point. Fifteen kidney tumors were used to microsatellite instability analysis.Alterations of microsatellite markers in the GaHV-2-infected samples were identified as differences in the electrophoretic migration or loss of major band(s) in comparison with normal muscular tissue samples DNA . MSI wasThe frequency of MSI for each marker is shown in Since the initial description of MSI in HNPCC in 1993, MSI has been identified in a wide variety of human cancers, both familial and sporadic MSI has been thought to be closely related to mutation of proteins involved in the MMR system, which normally maintains a low rate of spontaneous mutations and corrects replication errors. Many studies noted the phenomenon of mutation of mismatch repair genes in the development and progression of human tumors A panel of five microsatellites has been validated and recommended as a reference panel for future research in the field at a National Cancer Institute Workshop meeting In conclusion, this study demonstrates that the phenomenon of MSI does occur in Marek’s disease lymphoma induced by GaHV-2. Future studies will investigate the relationships among MMR, MSI and MD tumorigenesis to better understand the molecular nature of host genomic instability and virus infection.


Cichlidogyrus and Scutogyrus monogenean parasites, to uncover the presence of host-parasite cospeciation and to assess the level of morphological adaptation in parasites. This required the following steps, each one representing specific objectives of this paper: (1) to build phylogenetic trees for Cichlidogyrus and Scutogyrus species based on ribosomal DNA sequences, (2) to investigate phylogenetic relationships within West African cichlid fish based on the analysis of mitochondrial cytochrome b DNA sequences, (3) to investigate host-parasite cophylogenetic history to gain clues on parasite speciation process, and (4) to investigate the link between the morphology of the attachment apparatus and parasite phylogeny. Phylogenetic analyses supported the monophyletic origin of the Cichlidogyrus/Scutogyrus group, and suggested that Cichlidogyrus is polyphyletic and that Scutogyrus is monophyletic. The phylogeny of Cichlidae supported the separation of mouthbrooders and substrate-brooders and is consistent with the hypothesis that the mouthbrooding behavior of Oreochromis and Sarotherodon evolved from substrate-brooding behavior. The mapping of morphological characters of the haptor onto the parasite phylogenetic tree suggests that the attachment organ has evolved from a very simple form to a more complex one. The cophylogenetic analyses indicated a significant fit between trees using distance-based tests, but no significant cospeciation signal using tree-based tests, suggesting the presence of parasite duplications and host switches on related host species. This shed some light on the diversification process of Cichlidogyrus species parasitizing West African cichlids.The goals of this paper were to investigate phylogenetic and evolutionary patterns of cichlid fish from West Africa and their  The evolution of African cichlid fish is one of the most dramatic examples of extensive radiation and diversification in animals, reflected in a high number of studies Cichlids occur in Africa, Madagascar, Asia and the Neotropics. Their current distribution can be explained by two main hypotheses based on vicariance or dispersal model Dactylogyrus gill parasites from freshwater Cyprinidae Lamellodiscus gill parasites from marine Sparidae Gyrodactylus parasitizing many freshwater and marine fish species Polystoma parasitizing frogs Among metazoan parasites of cichlids, Monogenea are characterized by high species richness. In general, monogeneans have a direct life cycle and exhibit a high degree of morphological variability and species diversity. Moreover, they are highly host-specific compared to other groups of parasites Cichlidogyrus Paperna, 1960, Scutogyrus Pariselle & Euzet, 1995, Onchobdella Paperna, 1968, Enterogyrus Paperna, 1963 and Urogyrus Bilong-Bilong, Birgi & Euzet, 1994. While Enterogyrus and Urogyrus are mesoparasitic monogeneans of cichlids, Cichlidogyrus, Scutogyrus and Onchobdella are gill ectoparasites. Cichlidogyrus is the most diverse genus of monogeneans parasitizing cichlid fish, which are distributed among a wide range of fish species (more than 40 species within 11 genera) Scutogyrus and Onchobdella are restricted to several cichlid species; more precisely, Scutogyrus is restricted to Sarotherodon and Oreochromis, and Onchobdella to Hemichromis, Chromidotilapia and Pelmatochromis. Following Pariselle and Euzet Cichlidogyrus species, 6 Scutogyrus species, 8 Onchobdella species and 8 Enterogyrus species were described in cichlid fish from Africa, the Levant and Madagascar, among them 38 monogenean species  were reported in cichlid fish of West Africa and 22 species were revised in Senegal. Many Cichlidogyrus and Scutogyrus species are host-specific, i.e. from a total of 54 Cichlidogyrus and Scutogyrus species infesting West African cichlids, 36 species infest only a single cichlid species and 18 species infest two or more cichlid species Cichlidogyrus groups on Tilapia, Oreochromis and SarotherodonAfrican cichlids are parasitized by five genera of monogeneans belonging to the Dactylogyridea, Cichlidogyrus and Scutogyrus species, Pouyaud et al. Cichlidogyrus and Scutogyrus into four groups: “halli”, “scutogyrus”, “tiberianus” and “tilapiae” , and species infesting only the substrate brooder cichlids (genus Tilapia). Generally, indeed, a given species of Cichlidogyrus or Scutogyrus does not infect both mouthbrooders and substrate-brooders.Monogenean species determination is generally carried out using morphology and size of sclerotized parts of the attachment apparatus (termed haptor) and reproductive organs. Morphological characters have also been used to infer phylogenetic relationships between monogenean species. Concerning Cichlidogyrus and Scutogyrus species parasitizing cichlid fish in West Africa based on ribosomal DNA sequences, and phylogenetic analyses of Cichlidae to clarify the phylogenetic relationships among the cichlid fish living in West Africa using mitochondrial cytochrome b DNA sequences, and to investigate speciation processes in cichlid specific monogeneans using cophylogenetic analyses. In addition, parasite morphological characters were mapped onto parasite phylogeny to study the structural evolution of the haptor that could be related to adaptation to the host, and speciation processes.In this paper, we aimed to clarify these points using molecular phylogenetic trees for cichlid fish and their parasites, in order to study the evolution of feeding behavior in fish and morphology in parasites from independent evidence, i.e. molecular data. The objectives of this study were then to perform phylogenetic analyses of Cichlidogyrus species, 3 Scutogyrus species and 7 remaining species as outgroup . LSU sequences of Cichlidogyrus and Scutogyrus species varied from 637 bp (C. ergensi) to 844 bp (C. arthracanthus). No variability was observed among the individuals of C. tilapiae found in four different host species, i.e. H. fasciatus, O. niloticus, S. galilaeus and T. guineensis, and between Cichlidogyrus sp. 2 from S. galilaeus and T. guineensis. However, some nucleotide variability (i.e. p-distance corresponding to 0.037) was found between the individuals of C. halli 1 found in S. galilaeus and T. guineensis, and C. halli 2 found in O. niloticus. An unambiguous alignment spanned 533 positions. Information on the LSU rDNA alignment, as well the model selected by ModelTest and its parameters, are shown in The partial LSU rDNA sequences included 20 Cichlidogyrus with Scutogyrus species (i.e. Cichlidogyrus/Scutogyrus group) parasitizing Cichlidae formed a strongly supported monophyletic group, with Cichlidogyrus pouyaudi in basal position relative to other Cichlidogyrus and Scutogyrus species, which was strongly supported by bootstrap values  and moderately supported by Bayesian posterior probabilities.Based on the analyses of LSU rDNA sequences, Cichlidogyrus and Scutogyrus species. Partial SSU rDNA sequences varied from 423 bp  to 483 bp , and ITS1 sequences ranged between 330 bp  and 498 bp (C. pouyaudi). The partition homogeneity test implemented in PAUP*4b10 was used to test the congruence of the phylogenetic signal in partial SSU rDNA and ITS1 alignment. No significant difference was found (p = 0.1), sequences were combined, and the concatenated alignment spanned 542 positions . The position of Cichlidogyrus tilapiae parasitizing four different cichlid species . In ML reconstructions, the use of the codon model produced a tree with a low resolution . We then kept the well-resolved tree obtained using the codon partition scheme .For parasites, only the tree based on SSU rDNA and ITS1 sequence data  was usedUsing different cost schemes in Jane generated different results , but theCichlidogyrus. From this ancestral state, two different character states of marginal hooks may have derived (representing character states 2.1 in Cichlidogyrus and 2.3 found in Cichlidogyrus and Scutogyrus). Mapping also suggests the occurrence of two independent changes in the shape of marginal hook toward a state where all pairs of hooks are large (character state 2.4), one in Cichlidogyrus sp. 1 parasitizing Hemichromis letourneuxi and the other in Cichlidogyrus arthracanthus parasitizing Tilapia guineensis. The ancestral state of the ventral transverse bar  in Scutogyrus species. We could not identify the ancestral state of the dorsal transverse bar  and Protogyrodactylus  compared to gill monogeneans Cichlidogyrus and ScutogyrusScutogyrus was supported by phylogenetic analyses of ribosomal DNA sequences Scutogyrus genus arose from Cichlidogyrus, according to the morphology of dorsal and ventral transverse bars Scutogyrus species form a monophyletic group contrary to Wu et al. Cichlidogyrus, suggesting the need for a taxonomical revision of this genus.Based on phylogenetic analyses of LSU rDNA using as outgroup specific gill parasites of Cichlidogyrus pouyaudi parasitizing Tylochromis intermedius, which suggests that this parasite diverged earlier than the other Cichlidogyrus and Scutogyrus species. Cichlidogyrus pouyaudi was originally described from Tylochromis jentinki in West Africa Cichlidogyrus species. Pariselle and Euzet Cichlidogyrus species living on ancient cichlid fish such as Tylochromis species.The phylogenetic analyses using LSU rDNA sequences performed in this study placed in basal position Cichlidogyrus and Scutogyrus were identified using phylogenetic analyses based on SSU and ITS1. Two clades only contain strictly host specific Cichlidogyrus parasites, i.e. clade 1 parasitizing Hemichromis species and clade 4 parasitizing Tilapia guineensis. Contradictory to the prediction of Pouyaud et al. Cichlidogyrus species parasitized both mouthbrooders and substrate-brooders. Clade 2 included Cichlidogyrus parasitizing mouthbrooder cichlids (i.e. Oreochromis and Sarotherodon), except Cichlidogyrus sp. 2 found on the mouthbrooder Sarotherodon galilaeus as well as the substrate-brooder Tilapia guineensis. The absence of other Cichlidogyrus parasitizing Tilapia guineensis in this clade suggests a secondary host transfer of Cichlidogyrus sp. 2 from mouthbrooders to substrate-brooders. Cichlidogyrus thurstonae and C. douellouae, both parasites of mouthbrooders (in clade 5 of phylogenetic trees), probably colonized their mouthbrooder host species through lateral transfers (i.e. host switch), as suggested Pouyaud et al. Cichlidogyrus species infecting two cichlid groups with different reproductive behavioral strategies spans a broader geographical area . Concerning the generalist Cichlidogyrus species reported in our study, prevalence and abundance of C. halli 1 and Cichlidogyrus sp. 2 were higher in Sarotherodon galilaeus than in Tilapia guineensis, and higher prevalence and abundance were observed for C. tilapiae in Oreochromis niloticus than in Sarotherodon galilaeus, Tilapia guineensis and Hemichromis fasciatus. This suggests that generalist monogeneans display a level of host preference, i.e. a generalist selects preferentially one host species within its host range, such as a “common” host species compared to “additional” host species Cichlidogyrus in clade 2 as well as a mouthbrooder origin for the C. halli clade.In this study, six clades of gill parasites within b sequences supported three monophyletic groups of African cichlid species: substrate-brooders Hemichromis, mouthbrooders Sarotherodon and Oreochromis, and substrate-brooder Tilapia. The third group contains East African cichlid species. This separation of Sarotherodon and Oreochromis from Tilapia species has been previously reported based on the mitochondrial tRNAPro gene and the control region sequences Oreochromis and Sarotherodon genera evolved from a substrate-brooding behavior Tylochromis and Hemichromis diverged from the common cichlid stock first and then followed the divergence of Tilapia and Oreochromis. The separation of mouthbrooders and substrate-brooders is then supported by our phylogenetic analyses based on cytochrome b. Following Pouyaud et al. Scutogyrus parasitizes only mouthbrooders (Sarotherodon and Oreochromis). Further, Hemichromis species possess specific gill monogeneans from the Onchobdella genus, not shared by other cichlid species. However, some Cichlidogyrus species are able to parasitize both mouthbrooders and substrate-brooder cichlid species (see above). In the present study Tylochromis intermedius was found at a basal position relative to the other African cichlids. This basal position of Tylochromis among African cichlids supports the observation of Streelman et al. Tmo-4C4, and of Zardoya et al. Tylochromis as a sister group to African tilapiines The cichlid phylogeny based on cytochrome Cichlidogyrus/Scutogyrus parasites, “halli”, “scutogyrus”, “tiberianus” and “tilapiae”, using cluster analysis on morphometrical data from haptoral sclerotized parts. Our phylogenetic analyses did not support the monophyly of the “tiberianus” or “tilapiae” groups, because the most diversified clade 5 in our phylogenetic reconstructions included all species classified as “tiberianus” by Pouyaud et al. C. cubitus classified as “tilapiae”. The mapping of the haptor morphological characters performed in the present study relies on the hypothesis that C. pouyaudi diverged early compared to the other Cichlidogyrus and Scutogyrus species (see above) and thus, the characters of its haptor  are considered to form the ancestral Cichlidogyrus haptor type. Moreover, following previous studies on cichlid phylogenies (see above) and Pouyaud et al. Cichlidogyrus from Tilapia, Oreochromis and Sarotherodon compared to Cichlidogyrus of Hemichromis and Tylochromis . The mapping of structural characters in Cichlidogyrus and Scutogyrus species suggests that the haptor evolved from the simplest type toward the more complex. It also suggests a trend towards a clade-specific morphology with respect to marginal hooks, even if a few changes toward more complicated characters and one reversion to the ancestral state (for C. cubitus) were inferred in this analysis. Nevertheless, mapping does not support a different evolution of structural parts of the haptor in mouthbrooder and substrate-brooder cichlids and thus, it does not suggest any morphological adaptation of Cichlidogyrus species to the cichlids displaying different reproductive strategies. However, Cichlidogyrus/Scutogyrus species of only six cichlid fish species were considered here, and further studies (e.g. taking into account parasites from congeneric Tilapia or Sarotherodon host species) are needed to confirm these hypotheses. Pouyaud et al. Cichlidogyrus/Scutogyrus parasites, with a phylogeny reconstructed from ribosomal DNA sequences. The two dendrograms were different and only the dendrogram computed from haptor data was congruent with the phylogenetic tree. This suggests that the morphology of the haptor is more suitable for inferring phylogenetic relationships than the morphology of reproductive organs, maybe due to a faster rate of evolutionary change in the morphology of reproductive structures.Pouyaud et al. LamellodiscusGyrodactylusPolystomaDactylogyrusDactylogyrus diversification was explained in a large part by intrahost speciation (parasite duplication). All these studies suggest that the high host specificity of monogeneans is not linked to cospeciation. In the present study, distance-based analysis suggests that the global cophylogenetic structure in the Cichlidogyrus/Scutogyrus-cichlid system is significant. Tree-based analyses, however, indicate that this global structure is not significant, unless if the cost of host-switching is high. That supports previous hypotheses that host-switching, followed by speciation which results in the maintenance of high host specificity, is an important component of monogenean diversification. In all reconstructions, the number of duplications is high, which is also coherent with previously published hypotheses on monogenean evolution. Duplication is then suggested to be the main coevolutionary event explaining the diversification of gill monogeneans living on West African cichlid fish. This has been observed in other gill monogeneans-freshwater fish systems such as Dactylogyrus-Cyprinidae Thaparocleidus-Pangasidae (our unpublished data). The fact that global fit is significant with distance-based analysis only suggests that parasites switch to not too distantly related hosts (but not necessarily sister-species). This is supported by the fact that only few individual associations  were collected from the gills and stomachs of cichlid species , were caught in the Gambie River , in the Niokolo River , in the Mare de Simenti  and Mare de Wouring  and used for this study. Sarotherodon galilaeus individuals from Ivory Coast were also investigated for Scutogyrus species  but none were found during this study.A total of 28 parasite species belonging to four dactylogyridean genera , 1x PCR buffer, 1.50 mM MgCl2, 0.2 mM of dNTP, 0.50 µM of each primer, 0.1 mg/ml BSA and an aliquot of 30 ng of genomic DNA in a total volume of 30 µl. The polymerase chain reaction (PCR) was carried out using the following steps: 2 min at 94°C followed by 39 cycles of 20 sec at 94°C, 30 sec at 56°C and 1 min 30 sec at 72°C, and then 10 min of final elongation at 72°C. The partial SSU rDNA region and the entire ITS1 were amplified in one round using S1 and IR8 primers Taq polymerase (Fermentas), 1x PCR buffer, 1.50 mM MgCl2, 0.2 mM of dNTP, 0.8 µM of each primer, 0.1 mg/ml BSA and an aliquot of 30 ng of genomic DNA in a total volume of 30 µl. PCR was carried out in the Mastercycler ep gradient S (Eppendorf) with the following steps: 2 min at 94°C followed by 39 cycles of 1 min at 94°C, 1 min at 53°C and 1 min 30 sec at 72°C, and 10 min of final elongation at 72°C.Parasites were removed from ethanol and dried, and genomic DNA was extracted using DNeasy™ Tissue Kit (QIAGEN) following the manufacturer's instructions. The LSU rDNA region was amplified using C1 and D2 primers The PCR products were electrophoresed on a 1% agarose gel and then purified by either Wizard® SV Gel and PCR Clean-Up System (PROMEGA) or QIAquick PCR Purification Kit (QIAGEN). Sequencing was performed on an ABI 3130 Genetic Analyzer (Applied Biosystems) using Big Dye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems). Sequences were analyzed using Sequencher software (Gene Codes Corp) and deposited in EMBL under Accession numbers see .Cichlidogyrus and Scutogyrus as ingroup and Enterogyrus, Onchobdella and Protogyrodactylus as outgroup , maximum parsimony (MP) and maximum likelihood (ML) were performed in PAUP*4b10 The phylogeny of cichlid fishes was previously investigated using mitochondrial DNA sequences b (434 bp) of Tilapia guineensis, Hemichromis fasciatus, H. letourneuxi, Oreochromis niloticus and Sarotherodon galilaeus was amplified using forward primer L14725 (5′-CGAAGCTTGATATGAAAAACCATCGTTG-3′) designed by Farias et al. b (452 bp) of Tylochromis intermedius was amplified using forward primer (5′-TTTTACCAGGACTCTAACCAGGA-3′) and reverse primer (5′-GCYCCTCARAATGATATTTGTCC-3′), both of them designed in this study. The PCR reaction mixture consisted of 1.5 units of Taq polymerase (Fermentas), 1x PCR buffer, 2.50 mM MgCl2, 0.2 mM of dNTP, 0.3 µM of each primer and an aliquot of 30 ng of genomic DNA in a total volume of 30 µl. The PCR was carried out in the Mastercycler ep gradient S (Eppendorf) with the following steps: 4 min at 95°C followed by 30 cycles of 30 sec at 95°C, 30 sec at 50°C and 45 sec at 72°C, and 10 min of final elongation at 72°C. Electrophoresis was performed on 1% agarose gel and the PCR product was purified by Wizard® SV Gel and PCR Clean-Up System (PROMEGA) or QIAquick PCR Purification Kit (QIAGEN). Sequencing was performed on an ABI 3130 Genetic Analyzer (Applied Biosystems) using Big Dye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems). The sequences of cytochrome b were deposited in EMBL under Accession numbers  following the manufacturer's instructions. The partial region of cytochrome  numbers . DNA seqb sequences including 24 fish species as ingroup , and Cymatogaster, Etheostoma and Halichoeres as outgroup , no insertion or deletion event nor trimming was needed to improve the alignment, that was carefully checked visually. The list of fish species and accession numbers for cytochrome b sequences are shown in b DNA sequences were considered with an evolutionary model designed for coding sequences taking the genetic code into account Phylogenetic analyses were based on partial cytochrome Two methods of coevolutionary analyses were used: a distance-based method called ParaFit Distance-based methods focus only on the fit between host and parasite distances and do not test for the presence of any coevolutionary events. These methods use distance matrices and host associations to determine if hosts and parasites are randomly associated. An advantage of this is that they do not require fully resolved phylogenetic trees and can account for parasites associated with multiple hosts. In this study, patristic distances were calculated in CopyCat for each host and parasite phylogeny. The global fit between trees is computed and tested by randomizing individual host-parasite associations (links). ParaFit was also used to test whether a particular host-parasite link contributed to this global fit. Tests of significance were performed using 999 permutations.Tree-based methods use topologies and branch lengths to assess the fit between host and parasite phylogenies. These methods attempt to reconstruct the shared evolutionary history between hosts and their parasites with the smallest “cost” or smallest number of hypothesized historical events. A disadvantage of tree-based methods is that they require fully resolved phylogenies, and then do not account for phylogenetic uncertainty. Some of them, as TreeMap 1.0, do not appropriately account for parasites associated with multiple hosts in certain cases and therefore may underestimate host switching We investigated whether morphological evolution, i.e. evolution of the attachment apparatus, is linked to the parasite phylogeny. Categorical coding was used for character states, which were unordered because no relevant hypotheses could be applied for character polarization. Morphological characters of the haptor were mapped onto the fully resolved minimum evolution parasite tree inferred from the analysis of combined SSU rDNA and ITS1 data using MacClade version 4.0.1 with Farris optimization


The consumption of berry fruits, including strawberries, has been suggested to have beneficial effects against oxidative stress mediated diseases. Berries contain multiple phenolic compounds and secondary metabolites that contribute to their biological properties.ex vivo, and its cancer therapeutic and chemopreventive potential in mice models. Results of MTT, trypan blue and LDH assays suggested that MESB can induce cytotoxicity in cancer cells, irrespective of origin, in a concentration- and time-dependent manner. Treatment of mice bearing breast adenocarcinoma with MESB blocked the proliferation of tumor cells in a time-dependent manner and resulted in extended life span. Histological and immunohistochemical studies suggest that MESB treatment affected tumor cell proliferation by activating apoptosis and did not result in any side effects. Finally, we show that MESB can induce intrinsic pathway of apoptosis by activating p73 in breast cancer cells, when tumor suppressor gene p53 is mutated.Current study investigates the anticancer activity of the methanolic extract of strawberry (MESB) fruits in leukaemia (CEM) and breast cancer (T47D) cell lines The present study reveals that strawberry fruits possess both cancer preventive and therapeutic values and we discuss the mechanism by which it is achieved. A diet rich in fruits and vegetables has been associated with a reduced risk of diseases, such as cardiovascular disorders and cancer Previous studies suggest that consumption of berry fruits can have beneficial effects against diseases such as cancer Strawberries are a good source of natural antioxidants In most of the cancers, mutation in the tumor suppressor gene, p53, significantly contributes to cancer development Various phytochemicals and chemically synthesized small molecules induce apoptosis, largely through the activation of intrinsic pathway. Intrinsic apoptotic pathway involves a variety of stimuli from inside the cells like DNA damage, ROS generation etc. The major players of this pathway include BCL2 family of proteins, which are mainly classified as proapoptotic and antiapoptotic proteins, based on their activity. An imbalance in the ratio between these classes of proteins leads to damage of mitochondrial membrane integrity resulting in CYTOCHROME C release and CASPASE 9 followed by CASPASE 3 activation In the present study, we show that extracts prepared from Indian strawberry fruits induce cytotoxicity by activating intrinsic pathway of apoptosis, through a p53 independent mechanism in breast cancer cells. MESB also interferes with progression of tumors in breast cancer mouse models and results in the extended lifespan without affecting other cellular functions and body weight. Most importantly, we also provide evidence that strawberry consumption can delay tumorigenesis in mice.All chemicals used in the present study were of analytical grade and purchased from Sigma-Aldrich (USA) and antibodies were purchased from Santa Cruz Biotechnology (USA) and Cell Signalling Technology (USA).Indian strawberry fruits were purchased from the local markets, cut into small pieces and dried in shadow. The powdered strawberry was then extracted with methanol. Following evaporation, crude methanolic extracts were stored at room temperature under sterile conditions until further use.2.Human T-cell leukemia cells, CEM and human breast cancer cells, T47D were purchased from National Centre for Cell Science, Pune (India). Cells were cultured in RPMI 1640  containing 10% FBS , 100 U of Penicillin G/ml and 100 µg of streptomycin/ml  at 37°C in a humidified atmosphere containing 5% CO5 cells/ml) and increasing concentrations  of MESB was added and incubated. Cells were collected after 48 and 72 h of incubation. Number of viable cells was determined by trypan blue staining. Experiments were repeated three independent times and the data was presented as bar diagram with error bars.The effect of MESB on cell viability of CEM and T47D cells was determined by trypan blue dye exclusion assay 5 cells/ml) were treated with MESB  and incubated for 48 and 72 h. Cells were collected and subjected to MTT assay. Experiments were repeated three independent times, each with duplicate reactions and presented as bar diagram with error bars.The MTT assay was performed as described earlier Lactate dehydrogenase (LDH) assay was performed to assess the LDH release into the media following MESB treatment  on both CEM and T47D cells after 48 and 72 h of incubation as described earlier Cell lysate was prepared following treatment with MESB on T47D cells  as described and used for western blotting Western blotting studies were also performed using cytosolic extracts prepared following treatment with MESB. T47D cells were treated with MESB for 48 h . Cytosolic fractions were separated using mitochondrial extraction kit , western blotting was performed using anti-CYTOCHROME C and anti-SMAC/DIABLO.Mice were maintained as per the principles and guidelines of the ethical committee for animal care of Indian Institute of Science (IISc) in accordance with Indian National Law on animal care and use. The experimental design of the present study was approved by Institutional animal ethics committee (Ref. CAF/Ethics/125/2007/560), Indian Institute of Science, Bangalore, India.Swiss albino mice, 6–8 weeks old weighing approximately 18–22 g were purchased from central animal facility, Indian Institute of Science, Bangalore, India and maintained in the animal house, Department of Biochemistry, IISc. The animals were housed in polypropylene cages and provided standard pellet diet  and water ad libitum. The standard pellet diet composed of 21% protein, 5% lipids, 4% crude fiber, 8% ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2% vitamin and 55% nitrogen-free extract (carbohydrates). The mice were maintained under controlled temperature and humidity with a 12 h light/dark cycle.6 cells/22 g b. wt) were implanted into the peritoneal cavity of each donor mouse and allowed to multiply. The tumor cells were withdrawn, diluted in saline, counted and re-injected  to the right thigh of experimental animal for development of solid tumor as described Ehrlich ascites carcinoma (EAC) is an undifferentiated carcinoma, originally hyperdiploid and has high transplantable capability, rapid proliferation, shorter life span and 100% malignancy. EAC resembles human tumors which are most sensitive to chemotherapy due to the fact that they are undifferentiated and have a rapid growth rate. A fixed number of viable breast adenocarcinoma cells  control (n = 10). EAC injected animals were divided into 2 groups of 10 animals each. Group 2 was considered as tumor control and received no treatment. Group 3 received oral feeding of 2 g/kg of MESB dissolved in water after 12 days of tumor development and continued up to 45 days. The experiment was repeated 3 independent times.2, where ‘a’ and ‘b’ indicates the major and minor diameter, respectively th and 45th day of experimental period, one animal from each group  was sacrificed by cervical dislocation, tissues were collected and stored under appropriate conditions. Each experiment was repeated three independent times.Size of the developing tumor was measured in both group 2 and 3 animals by using vernier callipers on alternative days for the entire life span of the animals. Tumor volume was calculated using the formula V = 0.5abThe percentage of increase in lifespan was calculated and compared with control animals. The death pattern for control animals and MESB treated animals was recorded and % increase in lifespan was calculated using the formula [(T-C)/C] x100, where ‘T’ indicates the number of days the MESB treated animals survived and ‘C’ indicates the number of days tumor animals survived For each experiment, out of 10 animals, 5 (group 1) were orally fed with MESB (2 g/kg b.wt) for 20 days prior to the EAC injection and treatment was continued up to 45 days. Group 2 animals were considered as tumor control with no MESB treatment. The tumor volumes were measured after 12 days of EAC injection in both group 1 and group 2 animals. Each experiment was repeated two independent times. The percentage of increase in lifespan was calculated and compared with control animals.Tumor and liver tissues of normal and experimental mice were collected and processed as per standard protocol. Briefly, the tissues were embedded in paraffin wax, sectioned at 5–10 µm in rotary microtome  and stained with hematoxylin and eosin Swiss albino mice were fed with MESB (2 g/ml) for ten days to assess the side effects. Control and treated groups consisted of 8 mice each. Body weight was measured on every alternate day and the average body weight was plotted. To evaluate the effect of MESB on physiological functions, blood was collected after ten days of MESB treatment and analysed as described earlier 2O2 in PBS and IHC staining was performed as described earlier 2O2, counterstained with haematoxylin and mounted in DPX . Images were captured using light microscope .Immunohistochemical staining was performed on formalin fixed, paraffin embedded tissues, which were sectioned at a thickness of 5 µm. Slides were de-paraffinized using xylene, rehydrated and treated with 3% HValues are expressed as mean ± SEM for control and experimental samples and statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. For this analysis, Graphpad software prism 5.1 was used. The values were considered as statistically significant, if the p-value was equal to or less than 0.05.We have evaluated the cytotoxic effect of MESB on human breast cancer (T47D) and leukemia (CEM) cells using trypan blue dye exclusion and MTT assays. Results showed a dose-dependent effect on cell viability of CEM upon treatment with increasing concentrations of MESB . ComparaLDH assay was used to determine the cellular integrity following MESB treatment . Results showed a concentration and time-dependent increase in the LDH release upon treatment with MESB in both CEM and T47D cell lines .th day of tumor development on every alternate day until 45th day, 2 g/kg), a significant reduction in the tumor volume was observed compared to untreated control animals bearing tumor  compared to untreated controls . FurtherKi67 is a cell proliferation marker for tumor progression In order to understand the mechanism by which MESB induces cell death, we chose the breast cancer cell line, T47D, for further investigation. T47D cells were treated with increasing concentrations of MESB, cell extracts were prepared and used for immunoblotting analysis. Results showed activation of apoptotic marker, MCL-1, which acts as a proapoptotic protein upon cleavage. We find that MESB treatment resulted in prominent cleavage of MCL-1 as compared to the control . MESB trPreviously, it has been shown that the tumor suppressor gene, p53, is mutated in T47D cells p73 can induce apoptosis through both intrinsic as well as extrinsic pathways The correlation between higher intake of fruits, vegetables and decreased risk of developing certain cancers attributes to the content of antioxidants and other secondary metabolites present in plants. In the current study, we have evaluated the anticancer property of crude extract of strawberry fruits. Our studies using two cell lines of different origin suggest that irrespective of the cancer type, MESB could induce cytotoxicity, as shown by three independent assaying methods. We have used a mice model bearing breast adenocarcinoma to evaluate the chemotherapeutic and chemopreventive potential of strawberry. Besides, we have also identified the mechanism by which the strawberry extracts induce apoptosis in cancer cells.Previously, it has been reported that strawberry extracts induce cytotoxicity in microgram range Aronia melanocarpa juice also showed activation of p73 in p53 null cells p53 is a widely studied tumor suppressor gene. In response to DNA damage, p53 can act as a negative regulator for cell survival. It has been shown that most of the tumor malignancies are associated with mutation or allelic loss of p53 gene. In contrast, p73, a protein of p53 family, which shares significant structural and functional similarity with p53 protein, is rarely mutated in cancers MESB treatment on mice bearing tumor resulted in significant reduction in tumor volume without affecting the function of other organs along with approximately 4-fold increase in the lifespan. The histological evaluation showed that morphology and cellular architecture of the tissues was unaffected by the MESB treatment. Immunohistochemical studies also confirmed a decrease in cell proliferation as well as activation of apoptosis following treatment with MESB, suggesting regression of tumor in mice models. The observed protection from tumorigenesis upon pretreatment with strawberry extracts emphasizes the importance of strawberry fruits. Although the mechanism by which it exerts the chemoprevention is not clear, it reveals the added value of intake of strawberry fruits. Thus, the present study emphasizes the high therapeutic potential of strawberry. Further, our studies revealed that it can modulate the expression of p73, when p53 is mutated in cancers like breast cancer and can activate the mitochondrial pathway of apoptosis to abrogate cancer cell proliferation. Thus, our study clearly shows that strawberry can act as a good dietary, chemopreventive as well as therapeutic agent.


Aedes aegypti dispersion is the major reason for the increase in dengue transmission in South America. In Brazil, control of this mosquito strongly relies on the use of pyrethroids and organophosphates against adults and larvae, respectively. In consequence, many Ae. aegypti field populations are resistant to these compounds. Resistance has a significant adaptive value in the presence of insecticide treatment. However some selected mechanisms can influence important biological processes, leading to a high fitness cost in the absence of insecticide pressure. We investigated the dynamics of insecticide resistance and its potential fitness cost in five field populations and in a lineage selected for deltamethrin resistance in the laboratory, for nine generations. For all populations the life-trait parameters investigated were larval development, sex ratio, adult longevity, relative amount of ingested blood, rate of ovipositing females, size of egglaying and eggs viability. In the five natural populations, the effects on the life-trait parameters were discrete but directly proportional to resistance level. In addition, several viability parameters were strongly affected in the laboratory selected population compared to its unselected control. Our results suggest that mechanisms selected for organophosphate and pyrethroid resistance caused the accumulation of alleles with negative effects on different life-traits and corroborate the hypothesis that insecticide resistance is associated with a high fitness cost. Insecticide resistance is one of the major obstacles in the attempt to control disease vectors and agricultural pest insects. The continuous insecticide pressure can select insect populations with 1) an altered integument, less permissive to the insecticide penetration, 2) modifications in the structure or expression level of naturally detoxifying enzymes, 3) mutations in the insecticide target molecules, in the central nervous system, that hamper interaction with the insecticide, or 4) behavioral changes, such as avoiding contact with the insecticide Aedes aegypti specimens resistant to the pyrethroid permethrin In the case of insect vectors, insecticide resistance can interfere with their vectorial capacity, a condition estimated through biological (development and reproduction), ecological and behavioral parameters Ae. aegypti pyrethroid resistance in the country point to both metabolic and target site resistance mechanisms Ae. aegypti populations exhibiting different levels of resistance to pyrethroids and organophosphates, a series of life-trait parameters related to their vectorial capacity, such as larval developmental time, sex ratio, adult male and female longevity, relative amount of blood ingested by females, number of egglaying females, number and viability of eggs. Additionally, we selected one population during nine generations with deltamethrin, the pyrethroid presently employed in the country for the control of adults. During this process, the same aforementioned parameters were followed. We showed that a series of deleterious effects were selected together with resistance in laboratory conditions, while only a discrete commitment was noted in field populations. The implications of these results to vector control are discussed.Brazil presents a growing number of dengue cases, mainly due to the low efficacy of actions to control its vector, based on the use of organophosphates and pyrethroids, respectively against larvae and adults Aedes aegypti adult females from Natal/RN were selected with 1.5 µg deltamethrin / bottle from F1  to F3 . Deltamethrin dosage was then increased to 3.0 µg/bottle. The independent S  and R  lineages followed the same tendency within each group  throughout generations. At the end of the selection process (F9), a total of respectively 79 and 11% of pupae were formed in S and R groups. There is a significant progressive delay in larval development of R lineages . In all cases, a decrease was more prominent for R group  of 26.0 and 15.6 in respectively R and S groups at the F9 generation. In contrast, RR95 of the original field population was 18.6 Temephos dose response bioassays revealed resistance ratios  and 6th days  after larvae hatching  presented commitment in pupation. Delay in development dynamics was also observed in the Lepidoptera Spodoptera exigua selected with the pyrethroid fenvalerate Cydia pomonella (Lepidoptera: Tortricidae) strains, selected in the laboratory with the pyrethroid deltamethrin or with the Insect Growth Regulator diflubenzuron Factors selected for resistance may present direct pleiotropic effects in one or few life-trait aspects. In fact alterations in different traits can be considered manifestations of the insect physiological commitment to face the challenge represented by insecticide exposure. For instance, time of development is a primary aspect of fitness in disseminating mosquito populations Anopheles gambiae and Anopheles stephensi resistant to γ-HCH and dieldrin Longevity reduction, mainly in females, was observed in the pyrethroid exposed (R) group along the selection procedure. This is a relevant issue, since adult female longevity, and its duration related to the extrinsic incubation period of a pathogen, is one major aspect of mosquito vectorial capacity. By contrast, shortening in the longevity seemed not to be generally associated with insecticide resistance in field populations. Accordingly, no differences in longevity were observed in some strains of the malaria vectors 9 generation, compared to both Rockefeller and S group. Interestingly, Corbel et al. An. gambiae, was inversely proportional to the permethrin dosage in treated nets.The blood meal size is certainly one key parameter regarding both vectorial capacity and general fitness, since since it influences at least two aspects in a directly proportional way: the load of parasites ingested and the number of eggs laid. A significant reduction of the relative amount of ingested blood was observed in the pyrethroid selected R lineages at the FAe. aegypti females from a field population resistant to permethrin was lower when compared to a susceptible field strain. Additionally, resistant females took longer to lay their eggs. Lower insemination rates observed for γ-HCH and dieldrin resistant An. gambiae and An. stephensi females were attributed to a lower activity of resistant males Reduction in the number of egglaying females was also noted for R group, suggesting either a lower fecundity rate or, alternatively, decreased egglaying ability. Mebrahtu et al. Spodoptera exiguaCydia pomonellaAe. aegyptiAe. aegypti derived from permethrin resistant specimens.Reduction of both number and viability of eggs laid by pyrethroid selected females (group R) was noted. In contrast to the laboratory selection, evaluation of natural F1 populations revealed that only females from Fortaleza, which were the most resistant to temephos, exhibited reduction in the number, but not in the viability of deposited eggs. There is a series of reports describing reduction in the number of eggs laid by insecticide resistant strains derived from controlled selection experiments. This is the case of fenvalerate resistant The accumulation of negative effects in the life-trait parameters here investigated  was evident in laboratory selected lineages but rare in natural populations. The deleterious pleitropic effects of resistance alleles might be reduced over time thanks to the selection of modifiers in natural populations subjected to pressure for insecticide resistance,. In addition, the linkage disequilibrium between hitch-hiked deleterious alleles and resistance genes will also tend to decay over time due to recombination. Neither case is very likely to have occurred in the selected lines in only 9 generations. Here we presented a study showing problems related to insecticide resistance in life-trait parameters in a laboratory selected lineage as well as in the F1 of natural populations. Since there are very few options of compounds available for using in the control of insect disease vectors, the knowledge of the biochemical/molecular nature of the deleterious alleles associated to insecticide resistance can be an important tool to investigate the life time of an insecticide.Ae. aegypti field populations, belonging to three Brazilian regions, with different status of susceptibility to organophosphates and pyrethroids, were used at F1 generation: Henrique Jorge-Fortaleza/CE, Maceió/AL, Uberaba/MG, Aparecida de Goiânia/GO and Cuiabá/MT. Controlled pyrethroid selection, in the laboratory, was performed with mosquitoes collected at Natal/RN, Northeast Brazil, in 2005. With exception of Henrique Jorge, a district of Fortaleza, chosen because of its high infestation rates, samples were obtained through collection of eggs with ovitraps installed to cover the whole municipalities, as described elsewhere The Rockefeller (Rock) strain was established in 1959 in the Rockefeller Institute  Ae. aegypti eggs to hatch during 24 hours, 500 larvae were transferred to plastic basins (27×19×7 cm) filled with 1 L of dechlorinated water. Larvae were fed with 0.5 g of cat food , added each third day. Larvae were kept in BOD incubators at 28±1°C. Under these conditions all larvae develop into pupae in 5–6 days. Pupae were transferred to carton cages (17 cm diameter×18 cm high) and resulting adults were fed ad libitum with 10% (w/v) sugar solution. Adults were kept under 25±2°C and 70±10% rh. Adult females were fed on anesthetized guinea pigs. Three days after the blood meal oviposition cups, covered internally with a filter paper and partially filled with water, were introduced in the cages to receive oviposition. Two days later oviposition cups were removed from the cages and eggs were stored for further use up to six months after drying.After allowing resistant) females (R group) were then transferred to carton cages, in three distinct lineages, named R1–R3. Control lineages (S1–S3) consisted of mosquitoes reared in the same conditions, except for the absence of insecticide (S group). Each lineage started with 200 females at every new generation and remained isolated from the others during the whole procedure. Selection was performed during nine uninterrupted generations. In all lineages and for each generation females were allowed to have one blood meal/ week during three weeks.Pyrethroid selection was performed exclusively with female adults, 3–5 days old, exposed to deltamethrin in impregnated glass bottles Adult resistance status was evaluated through qualitative bottle bioassays Monitoring of pyrethroid selected groups was performed with bottles impregnated with 1.5 ug deltamethrin at F3 generation and with 3.0 ug afterwards. At least three deltamethrin impregnated bottles were used for each assay and for each lineage.9 generation of the selected lineages. Briefly, larvae were exposed to a series of 7–9 insecticide concentrations, with four replicas per concentration and 20 larvae per replica. Bioassays for each lineage were performed four times in different days. Resistance ratios (RR95) were calculated by comparison with Rockefeller values, using probit analysis .Dose-response bioassays with the organophosphate temephos were performed according to Lima et al. The following assays were performed at least twice; Rockefeller mosquitoes, taken directly from the laboratory colony, were always assayed in parallel, as an internal control.th day after larva hatching was evaluated.Eggs were immersed in water during 24 hours to induce hatching. Duplicate samples of 200 larvae each were carefully transferred to plastic basins containing 1 liter of dechlorinated water and reared as detailed in item 4.2. Pupae were daily scored and transferred to cages. The cumulative rate of pupae formed up to the 6Numbers of females and males resulting from the assay described above were compared.ad libitum. Statistical comparisons of female or male survivorship were performed arbitrarily on the 30th and 60th days after adult emergence.Adult longevity was monitored in duplicate samples of 100 couples each, which were kept in carton cages, fed with sugar solution Five-day old adult females were weighed in an analytical balance  before or after feeding in anesthetized guinea pigs during 30 minutes. In each case three pools of 10 females were used. Results are expressed as the ratio between the weights on both conditions.Several parameters were considered: a) the ratio of egglaying females; b) the number of eggs per female and c) the egg hatching rate. Oviposition of adult females, three days after a blood meal, was individually stimulated, in 6 cm diameter Petri dishes, covered with a wet filter paper, as described elsewhere Lineages within each laboratory reared group S (S1–3) and R (R1–3) were compared by ANOVA, followed by Tukey's multiple comparison test. When there were no significant differences among lineages of each group, data from groups S or R were pooled. Comparisons among Rockefeller, R and S groups were also made through ANOVA, followed by Tukey's multiple comparison test, except for egg numbers, performed with the non-parametric Kruskal-Wallis test. Numbers of female and male adults were compared by Fisher's exact test.Except were indicated, similar tests were performed to compare the same parameters among field populations. Analyses were conducted with GraphPad Prism version 5.00 for Windows .Figure S1dynamics of Ae. aegypti pupae formation in R and S lineages and Rockefeller strain. Mean rate with standard error are shown.(TIF)Click here for additional data file.Figure S2Longevity of Ae. aegypti3, F6 and F9 generations. adults derived from selection with (R) or without (S) pyrethroid pressure in the laboratory in the F Mean rates with standard error of alive females (panels A–C) and males (panels D–F) are represented. Rockefeller strain was reared in parallel as an internal control.(TIF)Click here for additional data file.Figure S3Blood meal ingestion of Ae. aegypti adults resulted from Rock (green) S (blue) and R (red) groups. Mean and standard error are shown.(TIF)Click here for additional data file.Table S1Student t test comparison between Aedes aegypti pooled R and S groups survival after exposure to deltamethrin under laboratory selection.(DOCX)Click here for additional data file.Table S2Aedes aegypti sex ratio of S and R groups under laboratory conditions, throughout generations.(DOCX)Click here for additional data file.Table S3Number of eggs per Aedes aegypti S and R females in the course of selection with deltamethrin.(DOCX)Click here for additional data file.