The costs of mounting an immune response are reflected in the reproductive fitness of the mosquito Anopheles gambiae

We have used Anopheles gambiae , a major vector of malaria in Africa, to test the hypothesis that the operation of a surveillance or immune system against microorganisms and parasites can be costly to the reproductive success of the host. Blood-fed mosquitoes were challenged with an immune elicitor, lipopolysaccharide (LPS), and their resultant antimicrobial activity, accumulation of yolk protein in the ovary and egg production was monitored. Humoral activity against the Gram-positive bacterium Micrococcus luteus was induced by LPS injection in a dose-responsive manner. LPS treatment also caused a concomitant significant reduction in the accumulation of protein in ovaries 24 h after injection and in the production of eggs during the same gonotrophic cycle. Unlike immune stimulation, reduction in reproductive fitness was not dose responsive. Oral administration of LPS also significantly reduced ovarian protein content although we could not detect the presence of anti- M. luteus activity in the gut tissue by using an inhibition zone assay. These findings indicate that immune stimulation imposed reproductive fitness costs on mosquitoes.     

Nutrient limitation results in juvenile hormone-mediated resorption of previtellogenic ovarian follicles in mosquitoes

Juvenile hormone (JH) is a central hormonal regulator of previtellogenic development in female Aedes aegypti mosquitoes. JH levels are low at eclosion and increase during the first day after adult emergence. This initial rise in JH is essential for female reproductive maturation. After previtellogenic maturation is complete, the mosquito enters a ‘state-of-arrest’ during which JH synthesis continues at a slower pace and further ovary development is repressed until a blood meal is taken. By examining the relationships between juvenile hormone, follicular resorption and nutrition in A. aegypti, we were able to define a critical role of JH during the previtellogenic resting stage. The rate of follicular resorption in resting stage mosquitoes is dependent on nutritional quality. Feeding water alone caused the rate of follicular resorption to reach over 20% by day 7 after emergence. Conversely, feeding a 20% sucrose solution caused resorption to remain below 5% during the entire experimental period. Mosquitoes fed 3% sucrose show rates of resorption intermediate between water and 20% sucrose and only reached 10% by day 7 after emergence. Follicular resorption is related to JH levels. Ligated abdomens separated from a source of JH (the corpora allata) showed an increase in resorption comparable to similarly aged starved mosquitoes (16%). Resorption in ligated abdomens was reduced to 6% by application of methoprene. The application of methoprene was also sufficient to prevent resorption in intact mosquitoes starved for 48 h (14% starved vs. 4% starved with methoprene). Additionally, active caspases were localized to resorbing follicles indicating that an apoptotic cell-death mechanism is responsible for follicular resorption during the previtellogenic resting stage. Taken together, these results indicate that JH mediates reproductive trade-offs in resting stage mosquitoes in response to nutrition.     

Sequence of apoptosis and inflammatory necrosis within the formative ovulatory site of sheep follicles

The aim of this study was to define the temporal and spatial patterns of apoptosis, necrosis and inflammation within preovulatory ovine follicles. A gonadotrophin surge was induced in pro-oestrous ewes by GnRH, and isolated follicles were hemisected into apical and basal segments at 0, 10, 18 and 22 h (the time of ovulatory stigma development) after GnRH. Ovarian surface epithelial and granulosa cells were isolated and assessed by fluorescence microscopy for membrane phosphatidylserine translocation-annexin V (early-stage apoptosis), oligonucleosomal DNA nick endlabelling (advanced apoptosis), and nuclear propidium iodide incorporation (necrotic membrane disruption). Thecal shells were analysed for interstitial blood cells. Preovulatory follicles were also hemisected and subjected to electrophoretic DNA degradation analysis. Annexin V binding and in situ DNA fragmentation among ovarian surface epithelial and granulosa cells along the follicular apex were high 18 and 22 h after GnRH. Propidium iodide staining of apical ovarian surface and granulosa cells was apparent at 22 h. There was a coincident increase within the apical theca as the time of ovulation approached in extravasated leucocytes (18 and 22 h) and erythrocytes (22 h). Apoptotic DNA laddering and necrotic DNA smears within the follicular apex were evident on agarose gels at 18 and 22 h, respectively. In contrast, ovarian surface epithelium not associated with the ovulation site and the basal follicular wall were largely unafflicted. It is suggested that both modalities of cellular death, apoptosis and necrosis (with acute inflammation and vascular injury), contribute progressively to follicular stigma formation and ovarian rupture.     

Ovarian regression and apoptosis in the South American teleost Leporinus taeniatus Lütken (Characiformes, Anostomidae) from the São Francisco Basin

Involution and resorption of both postovulatory and atretic follicles were analysed in piau‐jejo Leporinus taeniatus (Characiformes, Anostomidae) in order to evaluate the role of apoptosis during ovarian regression. Histological and ultrastructural analyses showed hallmarks of apoptosis in the granulosa: aggregation of compacted chromatin against the nuclear envelope, cell shrinkage, surface blebbing, loss of cell adhesion and cell fragmentation into apoptotic bodies. Protein synthesis activity preceded the onset of the cell death. The breakdown of the basement membrane led to the detachment of the granulosa cells into the follicular lumen. TUNEL‐positive reactions were detected in in situ DNA fragmentation of granulosa of both postovulatory and atretic follicles. Apoptosis increased in a time‐dependent manner contributing to reduction of the follicular areas. The apoptotic index (per cent of apoptotic cells) of the granulosa increased in postovulatory follicles soon after spawning, then these follicles degenerated and only remnants were observed at 7 days. In contrast, the granulosa cells reabsorbed the yolk during follicular atresia and the apoptotic index increased only in the late stage of regression. The results indicated apoptosis as the major mechanism to rapidly eliminate postovulatory follicles and being an essential process in the ovarian regression after spawning.     

Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae

Endosymbiotic Wolbachia bacteria are potent modulators of pathogen infection and transmission in multiple naturally and artificially infected insect species, including important vectors of human pathogens. Anopheles mosquitoes are naturally uninfected with Wolbachia, and stable artificial infections have not yet succeeded in this genus. Recent techniques have enabled establishment of somatic Wolbachia infections in Anopheles. Here, we characterize somatic infections of two diverse Wolbachia strains (wMelPop and wAlbB) in Anopheles gambiae, the major vector of human malaria. After infection, wMelPop disseminates widely in the mosquito, infecting the fat body, head, sensory organs and other tissues but is notably absent from the midgut and ovaries. Wolbachia initially induces the mosquito immune system, coincident with initial clearing of the infection, but then suppresses expression of immune genes, coincident with Wolbachia replication in the mosquito. Both wMelPop and wAlbB significantly inhibit Plasmodium falciparum oocyst levels in the mosquito midgut. Although not virulent in non-bloodfed mosquitoes, wMelPop exhibits a novel phenotype and is extremely virulent for approximately 12-24 hours post-bloodmeal, after which surviving mosquitoes exhibit similar mortality trajectories to control mosquitoes. The data suggest that if stable transinfections act in a similar manner to somatic infections, Wolbachia could potentially be used as part of a strategy to control the Anopheles mosquitoes that transmit malaria.     

Apoptosis and ovarian function: novel perspectives from the teleosts

Apoptosis is a fundamental mechanism in follicular atresia and postovulatory regression in mammals, but its role in teleost ovarian function is currently unknown. This study tested the hypotheses that apoptosis mediates follicular atresia in teleosts and is inducible in vitro by incubation in serum-free conditions. Vitellogenic follicles from rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) were incubated overnight in serum-free medium and examined for apoptosis by 3'-end-labeling and/or TUNEL analysis. Primary, postovulatory, and oocytectomized vitellogenic trout follicles and atretic goldfish follicles were evaluated in similar fashion. Overall, goldfish follicles had lower levels of DNA fragmentation than trout follicles. The DNA fragmentation in atretic goldfish follicles was similar to that measured in healthy vitellogenic and prematurational follicles; DNA fragmentation did not change after incubation. In the trout, postovulatory and oocytectomized vitellogenic follicles showed significantly greater in vitro susceptibility to apoptosis than intact vitellogenic follicles, whereas primary follicles were least susceptible. The TUNEL analyses revealed that in trout vitellogenic follicles, more thecal/epithelial cells than granulosa cells showed fragmented DNA in vivo, but incubation (24 h) did not result in increased apoptosis in cells of either type. These results indicate that apoptosis is involved in normal ovarian growth and postovulatory regression in teleosts, but that it does not appear to be an early event in teleost follicular atresia.     

Ancillary follicle and secondary corpora lutea formation following exogenous gonadotropin treatment in the domestic cat and effect of passive transfer of gonadotropin-neutralizing antisera

A combination regimen of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) was used to stimulate ovarian follicular development in domestic cats. The rate of elimination of eCG from circulation was estimated, and, following follicular aspiration, the formation of ancillary follicles and secondary CL was characterized. The effect of gonadotropin-neutralizing antisera on the development of secondary ovarian structures, CL function and humoral immune responses also was evaluated. After intramuscular injection, initial serum eCG concentrations were variable, with the elimination half-life estimated at 39 to 55 h and eCG persisting in circulation for several days. Following follicular aspiration, queens formed CL equal to the number of aspirated follicles and exhibited a rapid increase in progesterone concentration but developed high numbers of ancillary follicles by 5 d post aspiration. By 15 d post aspiration, all ancillary follicles had luteinized to form secondary CL. Treatment with neutralizing antisera at the time of follicular aspiration slowed (P < 0.05) CL formation but did not decrease (P > 0.05) the number of ancillary follicles or secondary CL. Progesterone concentrations did not differ (P > 0.05) from control queens while secondary humoral immune responses to eCG were qualitatively similar between groups. In summary, eCG was eliminated slowly from cats following intramuscular injection and this persistence in circulation may have contributed to the development of ancillary follicles and secondary CL. However, the administration of neutralizing antisera at the time of follicular aspiration was ineffective in preventing the formation of these secondary ovarian structures.     

Apoptosis in the fat body tissue of the beetle Tenebrio molitor parasitised by Hymenolepis diminuta

Many insects experience a decrease in reproductive output when parasitised. We are investigating mechanisms underlying this fecundity reduction using the rat tapeworm, Hymenolepis diminuta infection of Tenebrio molitor beetles. These include an increase in the resorption of developing ovarian follicles and a decrease in fat body synthesis of vitellogenin. The latter is the direct effect of a molecule produced by the parasite. Here we report a study to determine whether vitellogenin synthesis and follicle resorption are the result of parasite-induced apoptosis in the respective tissues and whether the parasite molecule acts directly on the fat body by inducing apoptosis. In vivo, the number of fat body cell nuclei with chromatin condensation are significantly elevated in parasitised females at all days examined and peaked at day 7 post-infection. A TUNEL assay to detect DNA fragmentation confirmed these observations of apoptosis. However, when fat body from uninfected females was co-cultured with live metacestodes they did not cause cells to die by apoptosis, showing that the induction signal does not come directly from the parasite. The follicle resorption observed in the ovaries of infected beetles was not associated with apoptosis of the epithelial cells. The possibility of several mechanisms underlying fecundity reduction is discussed.     

Multiparametric study of atresia in ewe antral follicles: histology, flow cytometry, internucleosomal DNA fragmentation, and lysosomal enzyme activities in granulosa cells and follicular fluid

The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI-), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow cytometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3-6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size-differential behavior was found in free or membrane-bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles.     

Oogenesis from human somatic stem cells and a role of immune adaptation in premature ovarian failure

The central thesis is that, while embryonic oocytes originate from extra-ovarian sources, those generated during fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE). With the assistance of immune system-related cells, primitive granulosa and germ cells appear to originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly in order to be recognized as self and renewed later. With the onset of menarche, a periodical follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs are always available during the prime reproductive period. The periodical follicular renewal ceases between 35-40 years of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. Premature ovarian failure (POF) may result from premature termination of follicular renewal during adulthood, possibly due to the alteration of fetal follicular development during immune adaptation (idiopathic POF), or due to the alteration of the adult immune system by cytostatic chemotherapy. Factors responsible for the diminution of follicular renewal may be responsible for the aging of other tissues and the whole body in general. However, our recent research shows that OSE stem cells may produce new eggs in vitro, even when derived from ovaries lacking primary follicles. Consequently, their in vitro fertilization (IVF) and subsequent utilization of embryos for intrauterine implantation may represent a novel IVF approach for providing genetically related children to women with ovarian infertility, which is worthy of consideration and further exploration.     

Life and death of female gametes during oogenesis and folliculogenesis

The vertebrate ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated early in ontogeny and thereafter continuously throughout reproductive life. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells, and only a minute fraction of the surviving follicles successfully complete the path to ovulation. The balance between signals for cell death and survival determines the destiny of the follicles. An abnormally high rate of cell death followed by atresia can negatively affect fertility and eventually lead irreversibly to premature ovarian failure. In this review we provide a short overview of the role of programmed cell death in prenatal differentiation of the primordial germ cells and in postnatal folliculogenesis. We also discuss the issue of neo-oogenesis. Next, we highlight molecules involved in regulation of granulosa cell apoptosis. We further discuss the potential use of scores for apoptosis in granulosa cells and characteristics of follicular fluid as prognostic markers for predicting the outcome of assisted reproduction. Potential therapeutic strategies for combating premature ovarian failure are also addressed.     

Differential effects of indomethacin on the sheep ovary: Prostaglandin biosynthesis, intracellular calcium, apoptosis, and ovulation

Cells of the apical wall of the dominant follicle and contiguous ovarian surface epithelium become apoptotic with the approach of ovulation in the sheep. It was hypothesized that indomethacin, an established inhibitor of prostaglandin biosynthesis and ovulation, would protect apical ovarian cells from programmed death. The anovulatory potencies of two systemic doses of indomethacin (200 and 800 mg) were tested in gonadotropin-stimulated ewes. A complete blockade of ovulation occurred at the higher dose of indomethacin. Ovulation was not inhibited by 200 mg indomethacin. Both doses of drug suppressed follicular prostaglandin production below pregonadotropin levels. Immunofluorescence detection of digoxigenin end-labeled (fragmented) DNA was used as a marker of apoptosis among ovarian surface epithelial and granulosa cells recovered from the apical hemisphere of preovulatory ovine follicles. Cellular DNA fragmentation was averted in animals given 800 mg indomethacin, whereas apoptosis ensued after 200 mg. A sustained increase in cytosolic calcium is generally a prerequisite to apoptotic DNA fragmentation and cell death. Indeed, intracellular calcium, detected by fluorescence of fura-2, was elevated in ovarian cells of animals destined to ovulate (controls, 200 mg indomethacin) in comparison to (safeguarded) cells of anovulatory ewes (800 mg indomethacin). These observations provide circumstantial evidence that apical ovarian cell degeneration by calcium-mediated apoptosis is a determinant of follicular instability and rupture, but that these events are unrelated to the gonadotropin-induced rise in prostanoid production characteristic of preovulatory follicles.     

Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Ovarian follicular atresia is mediated by heterophagy,autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Determination of onset of apoptosis in granulosa cells of the preovulatory follicles in the bonnet monkey (Macaca radiata): correlation with mitogen-activated protein kinase activities

During reproductive life, only a selected few ovarian follicles mature and ovulate, while the vast majority of follicles undergo a degenerative process called atresia. Recent studies have indicated that follicular atresia is mediated through apoptosis of follicular granulosa cells. The objectives of the present study were to determine the time of onset of apoptosis in granulosa cells of preovulatory follicles and to evaluate the consequences of gonadotropin withdrawal on mitogen-activated protein (MAP) kinase activities. Bonnet monkeys (Macaca radiata) undergoing controlled ovarian stimulation cycles were utilized for stimulation of multiple follicles, and granulosa cells were retrieved from preovulatory follicles at 24, 48, 72, and 96 h after stopping gonadotropin treatment. Serum and follicular fluid estradiol concentrations were highest at 24 h but declined precipitously (P < 0.05) to reach the lowest concentrations at 96 h; however, progesterone concentrations during this period did not increase, indicating the absence of luteinization. Quantitative analysis of genomic DNA by 3'-end labeling revealed the presence of low-molecular-weight fragments from 48 h onward, but by agarose gel electrophoresis, DNA laddering could be visualized only after 72 h. Messenger RNA expression for Bax, caspase-2, and caspase-3 increased with the onset of apoptosis. Immunoblot analysis of MAP kinases in lysates of granulosa cells (48-72 h) indicated increased (P < 0.05) levels of phosphorylated extracellular response kinase-1 and -2, Jun N-terminal kinase (JNK)-1 and -2, and p38. However, in vitro kinase assay data indicated that only phospho-JNK and -p38 activities were higher at 72 h compared to 24 h. These results demonstrate that granulosa cells of preovulatory follicles undergo apoptosis and that increased activities of phospho-JNK and -p38 are correlated with apoptosis in the primate.     

Morphological and quantitative evaluation of the ovarian recrudescence in Nile tilapia (Oreochromis niloticus) after spawning in captivity

The Nile tilapia is one of the most important fish species for aquaculture worldwide. Understanding their reproductive biology is essential for improving their aquaculture methods. The morphological and quantitative dynamics of ovarian recrudescence of Oreochromis niloticus was studied for 21 days postspawning. To accomplish this, breeding females were kept in controlled conditions and ovarian samples were collected weekly for histological, ultrastructural and morphometric analyses. Ovarian follicle morphology revealed an intense synthesis activity of the follicular cells, which actively contributed to formation of the zona radiata and oocyte development following spawning. Recently spawned ovaries contained follicles at all developmental stages, but they were predominantly early primary growth (～42%) and full‐grown follicles (～20%). Remnants of spawning, postovulatory follicle complexes represented approximately 5% of the former ovarian follicles immediately after spawning, and less than 1% after 7 days. Atretic follicles accounted for approximately 2% of the follicles studied during the period. The stock of primary growth follicles was stable during ovarian recrudescence, indicating their availability for continuous recruitment. Only the frequency of full‐grown follicles significantly increased in the ovaries during recrudescence, representing approximately 35% of the follicles 21 days postspawning. The diameters of all follicles were significantly different between the periods analyzed. The ovaries' morphological characteristics, the maintenance of young follicles stocks and the gradual and significant increase in the proportion and diameter of full‐grown follicles showed a rapid ovarian recovery and follicular growth of O. niloticus, in 21 days at 29.5°C, necessary for the next spawning. J. Morphol. 275:348–356, 2014. © 2013 Wiley Periodicals, Inc.     

Evaluating the costs of mosquito resistance to malaria parasites

Costly resistance mechanisms have been cited as an explanation for the widespread occurrence of parasitic infections, yet few studies have examined these costs in detail. A malaria-mosquito model has been used to test this concept by making a comparison of the fitness of highly susceptible lines of mosquitoes with lines that are resistant to infection. Malaria infection is known to cause a decrease in fecundity and fertility of mosquitoes; resistant mosquitoes were thus predicted to be fitter than susceptible ones. Anopheles gambiae were selected for refractoriness/resistance or for increased susceptibility to infection by Plasmodium yoelii nigeriensis. Additional lines that acted as controls for inbreeding depression were raised in parallel but not exposed to selection pressure. Selections were made in triplicate so that founder effects could be detected. Resistance mechanisms that were selected included melanotic encapsulation of parasites within 24 h postinfection and the complete disappearance of parasites from the gut. Costs of immune surveillance were assessed after an uninfected feed, and costs of immune deployment were assessed after exposure to infection and to infection and additional stresses. Mosquito survivorship was unaffected by either resistance to infection or by an increased burden of infection when compared with low levels of infection. In most cases reproductive fitness was equally affected by refractoriness or by infection. Resistant mosquitoes did not gain a fitness advantage by eliminating the parasites. Costs were consistently associated with larval production and egg hatch rate but rarely attributed to changes in blood feeding and never to changes in mosquito size. No advantages appeared to be gained by the offspring of resistant mosquitoes. Furthermore, we were unable to select for refractoriness in groups of mosquitoes in which 100% or 50% of the population were exposed to infection every generation for 22 generations. Under these selection pressures, no population had become completely refractory and only one became more resistant. Variations in fitness relative to control lines in different groups were attributed to founder effects. Our conclusion from these findings is that refractoriness to malaria is as costly as tolerance of infection.     

Inducible peroxidases mediate nitration of anopheles midgut cells undergoing apoptosis in response to Plasmodium invasion

Plasmodium berghei invasion of Anopheles stephensi midgut cells causes severe damage, induces expression of nitric-oxide synthase, and leads to apoptosis. The present study indicates that invasion results in tyrosine nitration, catalyzed as a two-step reaction in which nitric-oxide synthase induction is followed by increased peroxidase activity. Ookinete invasion induced localized expression of peroxidase enzymes, which catalyzed protein nitration in vitro in the presence of nitrite and H(2)O(2). Histochemical stainings revealed that when a parasite migrates laterally and invades more than one cell, the pattern of induced peroxidase activity is similar to that observed for tyrosine nitration. In Anopheles gambiae, ookinete invasion elicited similar responses; it induced expression of 5 of the 16 peroxidase genes predicted by the genome sequence and decreased mRNA levels of one of them. One of these inducible peroxidases has a C-terminal oxidase domain homologous to the catalytic moiety of phagocyte NADPH oxidase and could provide high local levels of superoxide anion (O(2)), that when dismutated would generate the local increase in H(2)O(2) required for nitration. Chemically induced apoptosis of midgut cells also activated expression of four ookinete-induced peroxidase genes, suggesting their involvement in general apoptotic responses. The two-step nitration reaction provides a mechanism to precisely localize and circumscribe the toxic products generated by defense reactions involving nitration. The present study furthers our understanding of the biochemistry of midgut defense reactions to parasite invasion and how these may influence the efficiency of malaria transmission by anopheline mosquitoes.     

Can Anopheles gambiae be infected with Wolbachia pipientis? Insights from an in vitro system

Wolbachia pipientis are maternally inherited endosymbionts associated with cytoplasmic incompatibility, a potential mechanism to drive transgenic traits into Anopheles populations for malaria control. W. pipientis infections are common in many mosquito genera but have never been observed in any Anopheles species, leading to the hypothesis that Anopheles mosquitoes are incapable of harboring infection. We used an in vitro system to evaluate the ability of Anopheles gambiae cells to harbor diverse W. pipientis infections. We successfully established W. pipientis infections (strains wRi and wAlbB) in the immunocompetent Anopheles gambiae cell line Sua5B. Infection was confirmed by PCR, antibiotic curing, DNA sequencing, and direct observation using fluorescence in situ hybridization. The infections were maintained at high passage rates for >30 passages. Our results indicate that there is no intrinsic genetic block to W. pipientis infection in A. gambiae cells, suggesting that establishment of in vivo W. pipientis infections in Anopheles mosquitoes may be feasible.