Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar

Background

To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).

Methodology/Principal Findings

A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.

Conclusions

Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.     

Immunolocalization of arabinogalactan proteins (AGPs) in reproductive structures of an early-divergent angiosperm,Trithuria (Hydatellaceae)

Background and Aims Trithuria

is the sole genus of Hydatellaceae, a family of the early-divergent angiosperm lineage Nymphaeales (water-lilies). In this study different arabinogalactan protein (AGP) epitopes in T. submersa were evaluated in order to understand the diversity of these proteins and their functions in flowering plants.

Methods

Immunolabelling of different AGPs and pectin epitopes in reproductive structures of T. submersa at the stage of early seed development was achieved by immunofluorescence of specific antibodies.

Key Results

AGPs in Trithuria pistil tissues could be important as structural proteins and also as possible signalling molecules. Intense labelling was obtained with anti-AGP antibodies both in the anthers and in the intine wall, the latter associated with pollen tube emergence.

Conclusions

AGPs could play a significant role in Trithuria reproduction, due to their specific presence in the pollen tube pathway. The results agree with labellings obtained for Arabidopsis and confirms the importance of AGPs in angiosperm reproductive structures as essential structural components and probably important signalling molecules.     

Genome-wide identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis

Background

A long juvenile period between germination and flowering is a common characteristic among fruit trees, including Malus hupehensis (Pamp.) Rehd., which is an apple rootstock widely used in China. microRNAs (miRNAs) play an important role in the regulation of phase transition and reproductive growth processes.

Results

M. hupehensis RNA libraries, one adult and one juvenile phase, were constructed using tree leaves and underwent high-throughput sequencing. We identified 42 known miRNA families and 172 novel miRNAs. We also identified 127 targets for 25 known miRNA families and 168 targets for 35 unique novel miRNAs using degradome sequencing. The identified miRNA targets were categorized into 58 biological processes, and the 123 targets of known miRNAs were associated with phase transition processes. The KEGG analysis revealed that these targets were involved in starch and sucrose metabolism, and plant hormone signal transduction. Expression profiling of miRNAs and their targets indicated multiple regulatory functions in the phase transition. The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition. mdm-miR160 and miRNA393, which regulate genes involved in auxin signal transduction, could also be involved in controlling this process. The identification of known and novel miRNAs and their targets provides new information on this regulatory process in M. hupehensis, which will contribute to the understanding of miRNA functions during growth, phase transition and reproduction in woody fruit trees.

Conclusions

The combination of sRNA and degradome sequencing can be used to better illustrate the profiling of hormone-regulated miRNAs and miRNA targets involving complex regulatory networks, which will contribute to the understanding of miRNA functions during growth, phase transition and reproductive growth in perennial woody fruit trees.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1125) contains supplementary material, which is available to authorized users.     

Gender-specific costs of reproduction on vegetative growth and physiological performance in the dioecious shrub Corema album

Background and Aims

Reproductive costs imply trade-offs in resource distribution at the physiological level, expressed as changes in future growth and/or reproduction. In dioecious species, females generally endure higher reproductive effort, although this is not necessarily expressed through higher somatic costs, as compensatory mechanisms may foster resource uptake during reproduction.

Methods

To assess effects of reproductive allocation on vegetative growth and physiological response in terms of costs and compensation mechanisms, a manipulative experiment of inflorescence bud removal was carried out in the sexually dimorphic species Corema album. Over two consecutive growing seasons, vegetative growth patterns, water status and photochemical efficiency were measured to evaluate gender-related differences.

Key Results

Suppression of reproductive allocation resulted in a direct reduction in somatic costs of reproduction, expressed through changes in growth variables and plant physiological status. Inflorescence bud removal was related to an increase in shoot elongation and water potential in male and female plants. The response to inflorescence bud removal showed gender-related differences that were related to the moment of maximum reproductive effort in each sexual form: flowering in males and fruiting in females. Delayed costs of reproduction were found in both water status and growth variables, showing gender-related differences in resource storage and use.

Conclusions

Results are consistent with the existence of a trade-off between reproductive and vegetative biomass, indicating that reproduction and growth depend on the same resource pool. Gender-related morphological and physiological differences arise as a response to different reproductive resource requirements. Delayed somatic costs provide evidence of gender-related differences in resource allocation and storage. Adaptive differences between genders in C. album may arise through the development of mechanisms which compensate for the cost of reproduction.     

High variation in clonal vs. sexual reproduction in populations of the wild strawberry, Fragaria virginiana (Rosaceae)

Background and Aims

Many plants reproduce both clonally and sexually, and the balance between the two modes of reproduction will vary among populations. Clonal reproduction was characterized in three populations of the wild strawberry, Fragaria virginiana, to determine the extent that reproductive mode varied locally between sites. The study sites were fragmented woodlands in Cook County, Illinois, USA.

Methods

A total of 95 strawberry ramets were sampled from the three sites via transects. Ramets were mapped and genotyped at five variable microsatellite loci. The variability at these five loci was sufficient to assign plants to clones with high confidence, and the spatial pattern of genets was mapped at each site.

Key Results

A total of 27 distinct multilocus genotypes were identified. Of these, 18 genotypes were detected only once, with the remaining nine detected in multiple ramets. The largest clone was identified in 16 ramets. No genets were shared between sites, and each site exhibited markedly different clonal and sexual recruitment patterns, ranging from two non-overlapping and widespread genets to 19 distinct genets. Only one flowering genet was female; the remainder were hermaphrodites.

Conclusions

Local population history or fine-scale ecological differences can result in dramatically different reproductive patterns at small spatial scales. This finding may be fairly widespread among clonal plant species, and studies that aim to characterize reproductive modes in species capable of asexual reproduction need to evaluate reproductive modes in multiple populations and sites.Key words: Clonal structure, gynodioecy, Fragaria virginiana, microsatellites, population genetic structure     

Does the silver moss Bryum argenteum exhibit sex-specific patterns in vegetative growth rate,asexual fitness or prezygotic reproductive investment?

Background and Aims

Expected life history trade-offs associated with sex differences in reproductive investment are often undetected in seed plants, with the difficulty arising from logistical issues of conducting controlled experiments. By controlling genotype, age and resource status of individuals, a bryophyte was assessed for sex-specific and location-specific patterns of vegetative, asexual and sexual growth/reproduction across a regional scale.

Methods

Twelve genotypes (six male, six female) of the dioecious bryophyte Bryum argenteum were subcultured to remove environmental effects, regenerated asexually to replicate each genotype 16 times, and grown over a period of 92 d. Plants were assessed for growth rates, asexual and sexual reproductive traits, and allocation to above- and below-ground regenerative biomass.

Key Results

The degree of sexual versus asexual reproductive investment appears to be under genetic control, with three distinct ecotypes found in this study. Protonemal growth rate was positively correlated with asexual reproduction and sexual reproduction, whereas asexual reproduction was negatively correlated (appeared to trade-off) with vegetative growth (shoot production). No sex-specific trade-offs were detected. Female sex-expressing shoots were longer than males, but the sexes did not differ in growth traits, asexual traits, sexual induction times, or above- and below-ground biomass. Males, however, had much higher rates of inflorescence production than females, which translated into a significantly higher (24x) prezygotic investment for males relative to females.

Conclusions

Evidence for three distinct ecotypes is presented for a bryophyte based on regeneration traits. Prior to zygote production, the sexes of this bryophyte did not differ in vegetative growth traits but significantly differed in reproductive investment, with the latter differences potentially implicated in the strongly biased female sex ratio. The disparity between males and females for prezygotic reproductive investment is the highest known for bryophytes.     

Clonal growth is enhanced in the absence of a mating morph: a comparative study of fertile stylar polymorphic and sterile monomorphic populations of Nymphoides montana (Menyanthaceae)

Background and Aims

Many aquatic species with stylar polymorphisms have the capacity for clonal and sexual reproduction and are sensitive to the balance of the two reproductive modes when there are a limited number of mating morphs within a population. This study asked how the clonal and sexual reproductive modes perform in populations that contain only a single morph and where fitness gain through sexual reproduction is rare. In clonal aquatic Nymphoides montana, polymorphic populations normally contain two mating morphs in equal frequencies. Populations are sexually fertile and appear to be maintained by pollen transfer between the two partners. However, in a monomorphic population of N. montana where mating opportunities are unavailable, female and male function is impaired and clonality maintains the population. Here, the consequences of intraspecific variation in sexuality were explored between monomorphic and polymorphic N. montana populations in eastern Australia.

Methods

Comparative measurements of male and female fertility, total dry mass and genotypic diversity using ISSR markers were made between populations with variable sexuality.

Key Results and Conclusions

Very few seeds were produced in the monomorphic population under natural and glasshouse conditions due to dysfunctional pollen and ovules. Stigma–anther separation was minimal in the monomorphic population, which may be a consequence of the relaxed selective pressures that regulate the maintenance of sexual function. However, clonal reproduction was favoured at the expense of sexual reproduction in the monomorphic population; this may facilitate the establishment of sterility throughout the population via resource reallocation or pleiotropic effects. The ISSR results showed that the monomorphic population was one large, single genotype, unlike the multi-genotypic fertile polymorphic populations. Evolutionary loss of sex in a clonal population in which a mating morph is absent was evident; under these conditions clonal growth may assure reproduction and expand the population via spreading stolons.     

Differential competitive ability between sexes in the dioecious Antennaria dioica (Asteraceae)

Background and Aims

Differences in competitive ability between the sexes of dioecious plants are expected as a result of allocation trade-offs associated with sex-differential reproductive costs. However, the available data on competitive ability in dioecious plants are scarce and contradictory. In this study sexual competition was evaluated using the dioecious plant Antennaria dioica in a common garden transplantation experiment.

Methods

Male and female plants were grown for 3 years either in isolation, or in competition with a plant of the same sex or the opposite sex. Flowering phenology, sexual and asexual reproduction, plant growth, nutrient content and arbuscular mycorrhizal colonization in the roots were assessed.

Key Results

Our results showed little evidence of sexual differences in competitive ability. Both sexes suffered similarly from competition, and competitive effects were manifested in some traits related to fitness but not in others. Survival was unaffected by competition, but competing plants reduced their vegetative growth and reproductive investment compared with non-competing plants. In addition, differences in sexual competitive ability were observed in relation to flowering frequency, an important life history trait not reported in previous studies.

Conclusions

The findings indicate that female and male A. dioica plants possess similar intersexual competitive abilities which may be related to the similar costs of reproduction between sexes in this species. Nevertheless, intrasexual competition is higher in females, giving support for asymmetric niche segregation between the sexes.     

Limitations to reproductive output and genetic rescue in populations of the rare shrub Grevillea repens (Proteaceae)

Background and Aims

When conserving rare plant species, managers are often faced with small and/or isolated populations displaying low levels of sexual reproduction and genetic variation. One option for reinvigorating these populations is the introduction of genetic material from other sites, but in some cases fitness may be reduced as a result of outbreeding depression. Here the pollination biology of the rare shrub Grevillea repens is studied across its natural range and reproductive responses following cross-pollination among populations are examined to determine factors that may be limiting sexual reproduction and the potential for genetic rescue.

Methods

Pollen manipulation treatments (self-, autogamous self-, cross- and open pollination) were applied to flowers to examine the breeding system and fruit and seed production in five populations of G. repens. Pollen production, presentation and viability were investigated and interpopulation crosses of increasing genetic distance performed among the populations.

Key Results

The study species is self-incompatible and displayed very low natural seed set over two seasons, due partly to low pollen viability in one of the populations. Within-population crossing increased fruit and seed production at some sites, indicating pollinator limitation. Interpopulation crosses further increased reproductive output in one population, suggesting mate limitation, and for this site there was a positive relationship between genetic distance among populations and the size of genetic rescue benefits. However, in other populations there was a decrease in fruit and seed set with increasing genetic distance.

Conclusions

The results highlight that management strategies involving interpopulation crosses can improve reproductive output in small, isolated populations of rare plants, but guidelines need to be developed on a population by population basis.Key words: Grevillea repens, Proteaceae, genetic rescue, pollination ecology, self-incompatibility, breeding system, interpopulation cross, outbreeding depression, pollinator limitation, mate limitation, resource limitation     

Limited mate availability decreases reproductive success of fragmented populations of Linnaea borealis, a rare, clonal self-incompatible plant

Background and Aims

Small populations of rare plant species are increasingly reported to have high levels of reproductive failure. The objective of this study was to understand the principal constraints on sexual reproduction in small fragmented populations of a rare clonal self-incompatible plant.

Methods

The pollinator spectrum, diversity of flower colour, natural pollination and fruit-set levels of L. borealis were examined in Scotland. Artificially crossed seed production was compared within and between different flower colour types and patches.

Key Results

Linnaea borealis was pollinated by a diverse spectrum of insect species and the principal pollinators were muscid, syrphid and empid flies which mostly moved only small distances (<0·25 m) between flowers when foraging. Natural pollination levels were high, indicating high pollinator effectiveness, but fruit set was very low in most patches. Flower colour diversity was low in most patches and only those with a diversity of flower colour types had high fruiting success. Pollination experiments showed L. borealis to be highly self-incompatible and artificial crosses within and between patches and flower colour types confirmed that low fruit success was the result of a lack of compatible mates and limited pollen movement between them. Evidence of isolation from pollen exchange was apparent at as little as 6 m and severe at 30 m and beyond.

Conclusions

Limited mate availability and isolation from pollen exchange compromise the reproductive success of fragmented populations of L. borealis in Scotland. A diversity of compatible mates situated within close proximity (<6 m) is the key requirement to ensure high natural fruiting success. This study emphasizes that an understanding of the breeding system, pollinator spectrum and potential for interconnectivity via pollinator movement are fundamental to identify isolation distances and to establish when conservation intervention is necessary for rare species.Key words: Linnaea borealis, clonal, self-incompatible, reproductive failure, fragmented populations, isolation, pollination     

Cytotype stability, facultative apomixis and geographical parthenogenesis in Ranunculus kuepferi (Ranunculaceae)

Background and Aims

Asexual organisms are more widespread in previously glaciated areas than their sexual relatives (‘geographical parthenogenesis’). In plants, this pattern is probably dependent on reproductive isolation and stability of cytotypes within their respective distribution areas. Both partial apomixis and introgressive hybridization potentially destabilize the spatial separation of sexual and apomictic populations. The wide distribution of apomicts may be further enhanced by uniparental reproduction which is advantageous for colonization. These factors are studied in the alpine species Ranunculus kuepferi.

Methods

Geographical distribution, diversity and mode of reproduction of cytotypes were assessed using flow cytometry and flow cytometric seed screening on samples from 59 natural populations of Ranunculus kuepferi. Seed set of cytotypes was compared in the wild.

Key Results

Diploid sexuals are confined to the south-western parts of the Alps, while tetraploid apomicts dominate in previously glaciated and in geographically isolated areas despite a significantly lower fertility. Other cytotypes (3x, 5x and 6x) occur mainly in the sympatric zone, but without establishing populations. The tetraploids are predominantly apomictic, but also show a partial apomixis via an uncoupling of apomeiosis and parthenogenesis in the seed material. Both pseudogamy and autonomous endosperm formation are observed which may enhance uniparental reproduction.

Conclusions

Diploids occupy a glacial relic area and resist introgression of apomixis, probably because of a significantly higher seed set. Among the polyploids, only apomictic tetraploids form stable populations; the other cytotypes arising from partial apomixis fail to establish, probably because of minority cytotype disadvantages. Tetraploid apomicts colonize previously devastated and also distant areas via long-distance dispersal, confirming Baker''s law of an advantage of uniparental reproduction. It is concluded that stability of cytotypes and of modes of reproduction are important factors for establishing a pattern of geographical parthenogenesis.     

Differential costs of reproduction in females and hermaphrodites in a gynodioecious plant

Background and Aims

Plants exhibit a variety of reproductive systems where unisexual (females or males) morphs coexist with hermaphrodites. The maintenance of dimorphic and polymorphic reproductive systems may be problematic. For example, to coexist with hermaphrodites the females of gynodioecious species have to compensate for the lack of male function. In our study species, Geranium sylvaticum, a perennial gynodioecious herb, the relative seed fitness advantage of females varies significantly between years within populations as well as among populations. Differences in reproductive investment between females and hermaphrodites may lead to differences in future survival, growth and reproductive success, i.e. to differential costs of reproduction. Since females of this species produce more seeds, higher costs of reproduction in females than in hermaphrodites were expected. Due to the higher costs of reproduction, the yearly variation in reproductive output of females might be more pronounced than that of hermaphrodites.

Methods

Using supplemental hand-pollination of females and hermaphrodites of G. sylvaticum we examined if increased reproductive output leads to differential costs of reproduction in terms of survival, probability of flowering, and seed production in the following year.

Key Results

Experimentally increased reproductive output had differential effects on the reproduction of females and hermaphrodites. In hermaphrodites, the probability of flowering decreased significantly in the following year, whereas in females the costs were expressed in terms of decreased future seed production.

Conclusions

When combining the probability of flowering and seed production per plant to estimate the multiplicative change in fitness, female plants showed a 56 % and hermaphrodites showed a 39 % decrease in fitness due to experimentally increased reproduction. Therefore, in total, female plants seem to be more sensitive to the cost of reproduction in terms of seed fitness than hermaphrodites.     

Apomixis is not prevalent in subnival to nival plants of the European Alps

Background and Aims

High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic species occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown.

Methods

The mode of reproduction in 14 species belonging to seven families was investigated via flow cytometric seed screen. The sampling comprised 12 species typical for nival to subnival plant communities of the European Alps without any previous information on apomixis (Achillea atrata, Androsace alpina, Arabis caerulea, Erigeron uniflorus, Gnaphalium hoppeanum, Leucanthemopsis alpina, Oxyria digyna, Potentilla frigida, Ranunculus alpestris, R. glacialis, R. pygmaeus and Saxifraga bryoides), and two high-alpine species with apomixis reported from other geographical areas (Leontopodium alpinum and Potentilla crantzii).

Key Results

Flow cytometric data were clearly interpretable for all 46 population samples, confirming the utility of the method for broad screenings on non-model organisms. Formation of endosperm in all species of Asteraceae was documented. Ratios of endosperm : embryo showed pseudogamous apomixis for Potentilla crantzii (ratio approx. 3), but sexual reproduction for all other species (ratios approx. 1·5).

Conclusions

The occurrence of apomixis is not correlated to high altitudes, and cannot be readily explained by selective forces due to environmental conditions. The investigated species have probably other adaptations to high altitudes to maintain reproductive assurance via sexuality. We hypothesize that shifts to apomixis are rather connected to frequencies of polyploidization than to ecological conditions.     

Host range and selectivity of the hemiparasitic plant Thesium chinense (Santalaceae)

Background and Aims

Thesium chinense is a hemiparasitic plant that is common in grassland habitats of eastern Asia. Although the physiology of Thesium has been well studied in attempts to control its weedy habit, there have been few ecological investigations of its parasitic life history. Thesium chinense is thought to parasitize species of Poaceae, but evidence remains circumstantial.

Methods

A vegetation survey was conducted to test whether any plant species occurs significantly more often in plots with T. chinense than expected. In addition, haustorial connections were examined directly by excavating the roots and post-attachment host selectivity was evaluated by comparing the observed numbers of haustoria on different hosts against those expected according to the relative below-ground biomass. Haustorium sizes were also compared among host species.

Key Results

Only two of the 38 species recorded, Lespedeza juncea and Eragrostis curvula, occurred more often in plots with Thesium than expected. In contrast to this, T. chinense parasitized 22 plant species in 11 families, corresponding to 57·9 % of plant species found at the study site. Haustoria were non-randomly distributed among host species, suggesting that there is some post-attachment host selectivity. Thesium chinense generally preferred the Poaceae, although haustoria formed on the Fabaceae were larger than those on other hosts.

Conclusions

This is the first quantitative investigation of the host range and selectivity of hemiparasitic plants of the Santalales. The preference for Fabaceae as hosts may be linked to the greater nutrient availability in these nitrogen-fixing plants.Key words: Haustorium, hemiparasite, host range, host selectivity, Santalaceae, Thesium chinense     

Mirna expression profiles identify drivers in colorectal and pancreatic cancers

Background and Aim

Altered expression of microRNAs (miRNAs) hallmarks many cancer types. The study of the associations of miRNA expression profile and cancer phenotype could help identify the links between deregulation of miRNA expression and oncogenic pathways.

Methods

Expression profiling of 866 human miRNAs in 19 colorectal and 17 pancreatic cancers and in matched adjacent normal tissues was investigated. Classical paired t-test and random forest analyses were applied to identify miRNAs associated with tissue-specific tumors. Network analysis based on a computational approach to mine associations between cancer types and miRNAs was performed.

Results

The merge between the two statistical methods used to intersect the miRNAs differentially expressed in colon and pancreatic cancers allowed the identification of cancer-specific miRNA alterations. By miRNA-network analysis, tissue-specific patterns of miRNA deregulation were traced: the driving miRNAs were miR-195, miR-1280, miR-140-3p and miR-1246 in colorectal tumors, and miR-103, miR-23a and miR-15b in pancreatic cancers.

Conclusion

MiRNA expression profiles may identify cancer-specific signatures and potentially useful biomarkers for the diagnosis of tissue specific cancers. miRNA-network analysis help identify altered miRNA regulatory networks that could play a role in tumor pathogenesis.     

Modeling the Interruption of the Transmission of Soil-Transmitted Helminths by Repeated Mass Chemotherapy of School-Age Children

Background

The control or elimination of neglected tropical diseases has recently become the focus of increased interest and funding from international agencies through the donation of drugs. Resources are becoming available for the treatment of soil-transmitted helminth (STH) infection through school-based deworming strategies. However, little research has been conducted to assess the impact of STH treatment that could be used to guide the design of efficient elimination programs.

Methodology

We construct and analyse an age-structured model of STH population dynamics under regular treatment. We investigate the potential for elimination with finite rounds of treatment, and how this depends on the value of the basic reproductive number R₀ and treatment frequency.

Principal findings

Analysis of the model indicates that its behaviour is determined by key parameter groupings describing the basic reproduction number and the fraction of it attributable to the treated group, the timescale of material in the environment and the frequency and efficacy of treatment. Mechanisms of sexual reproduction and persistence of infectious material in the environment are found to be much more important in the context of elimination than in the undisturbed baseline scenario. For a given rate of drug use, sexual reproduction dictates that less frequent, higher coverage treatment is more effective. For a given treatment coverage level, the lifespan of infectious material in the environment places a limit on the effectiveness of increased treatment frequency.

Conclusions

Our work suggests that for models to capture the dynamics of parasite burdens in populations under regular treatment as elimination is approached, they need to include the effects of sexual reproduction among parasites and the dynamics infectious material in the reservoir. The interaction of these two mechanisms has a strong effect on optimum treatment strategies, both in terms of how frequently to treat and for how long.