Specialized host-plant performance of the cotton aphid is altered by experience

Although distinct host specialization is observed for the cotton-melon aphid (Aphis gossypii Glover) on cotton and cucurbit plants, it is still ambiguous whether the specialization is altered by experience on a novel host plant. Here the performance of cotton and cucurbit-specialized aphids, A. gossypii on novel host plants was studied by a host-selection test and by the life-table method. The two host-specialized aphids cannot survive and establish populations after reciprocal host transfers. They have ability to recognize the host plants on which they were reared, and escape behavior from novel hosts was observed. Interestingly, the cotton and cucurbit-specialized aphids survive and reproduce normally on hibiscus (Hibiscus syriacus), a main overwintering host plant, and host-fidelity of A. gossypii to cucurbit plants is altered by feeding and living experience on hibiscus, which confers the same capacity to use cotton and cucumber on to the cucurbit-specialized population, but host-fidelity to cotton is not altered and the fitness of the cotton specialized population to cucumber is still poorer. A. gossypii from hibiscus has a significant preference for cotton to cucumber in the host-selection process, and none stays on cucumber more than 20 h after transfer. The results presented imply that cucurbit-specialized aphids might not return to an overwintering host plant (hibiscus) in wild fields, so host conservatism to cucurbit plants is maintained. The potential of cucurbit-specialized aphids of A. gossypii to use cotton plants, intermediated by experience on hibiscus, suggests that the specialized host-plant performance of phytophagous insects is not wholly conservative.     

Identification of Aphis gossypii Glover (Hemiptera: Aphididae) Biotypes from Different Host Plants in North China

Background

The cotton-melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a polyphagous species with a worldwide distribution and a variety of biotypes. North China is a traditional agricultural area with abundant winter and summer hosts of A. gossypii. While the life cycles of A. gossypii on different plants have been well studied, those of the biotypes of North China are still unclear.

Results

Host transfer experiments showed that A. gossypii from North China has two host-specialized biotypes: cotton and cucumber. Based on complete mitochondrial sequences, we identified a molecular marker with five single-nucleotide polymorphisms to distinguish the biotypes. Using this marker, a large-scale study of biotypes on primary winter and summer hosts was conducted. All A. gossypii collected from three primary hosts—hibiscus, pomegranate, and Chinese prickly ash—were cotton biotypes, with more cotton-melon aphids found on hibiscus than the other two species. In May, alate cotton and cucumber biotypes coexisted on cotton and cucumber seedlings, but each preferred its natal host. Both biotypes existed on zucchini, although the cucumber biotype was more numerous. Aphids on muskmelon were all cucumber biotypes, whereas most aphids on kidney bean were cotton biotypes. Aphids on seedlings of potato and cowpea belong to other species. In August, aphids on cotton and cucumber were the respective biotypes, with zucchini still hosting both biotypes as before. Thus, the biotypes had different fitnesses on different host plants.

Conclusions

Two host-specialized biotypes (cotton and cucumber) are present in North China. Hibiscus, pomegranate, and Chinese prickly ash can serve as winter hosts for the cotton biotype but not the cucumber biotype in North China. The fitnesses of the two host-specialized biotypes differ on various summer hosts. When alate aphids migrate to summer hosts, they cannot accurately land on the corresponding plant.     

Interference competition between Aphidius matricariae and Praon volucre (Hymenoptera: Braconidae) attacking two common aphid species

Interference competition is a common phenomenon that occurs among adult females of different species to gain the highest food resources at the same time. In this research, extrinsic competition between Aphidius matricariae and Praon volucre on different densities and stages of two important pests of greenhouse crops in the world, Aphis gossypii and Myzus persicae, were investigated. The results of this study showed that total percentage parasitism of second, third and fourth nymphal instars of A. gossypii and M. persicae by A. matricariae, and P. volucre were affected by extrinsic competition. A combination of A. matricariae and P. volucre on A. gossypii had a negative effect on performance of these parasitoids. Although extrinsic competition reduced the efficiency of A. matricariae and P. volucre on M. persicae, total parasitism of M. persicae by A. matricariae and P. volucre (combination of two parasitoids) increased compared to A. matricariae or P. volucre acting alone. Also the results indicated that the extrinsic competition between A. matricariae and P. volucre on both A. gossypii and M. persicae was apparently more intense when presented with 25 hosts compared to 50 and 100 hosts. The results of this research show important information to determine an appropriate combination of these two parasitoid wasps for biological control of A. gossypii and M. persicae in greenhouses.     

Mechanisms of species‐sorting: effect of habitat occupancy on aphids' host plant selection

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Time allocation of the parasitoidAphidius colemani (Hymenoptera: Aphidiidae) foraging forAphis gossypii (Homoptera: Aphidae) on cucumber leaves

The time allocation of individualAphidius colemani female parasitoids foraging forAphis gossypii nymphs on cucumber leaves has been investigated. Apart from experiences on the current leaf (such as density of hosts on the current leaf, density of hosts on a neighboring leaf and encounters with hosts on the current leaf), the effect of previous leaf visits on the time allocation was studied. Behavioral records were analyzed by means of the proportional hazards model, to determine the tendency of leaving the current leaf. The leaving tendency decreased only on leaves with a high host density (100 aphids), thus increasing the giving up time since the latest encounter. Rejection of aphids had no influence on the leaving tendency. To assess the effect of the number of hosts encountered on the leaving tendency, we considered three classes: 0–30 encounters, 31–100 encounters, and 100 or more encounters with hosts. The effect of the number of hosts encountered differed at different aphid densities. When fewer than 10 aphids were present the leaving tendency was much greater after 30 encounters than beforehand. At a density of 100 aphids the leaving tendency was lower than at the other aphid densities and increased only after 100 encounters. The density of hosts on a neighboring leaf, ranging from 0 to 100 hosts, had a negligible effect on the leaving tendency. Repeated visits to leaves with 10 unparasitized aphids resulted in an increase in the leaving tendency after 10 visits. It is argued that the parasitoids have some innate expectancy of host availability and that they concentrate on high-density patches.     

Influence of vermicompost and cucumber cultivar on population growth of Aphis gossypii Glover

The melon aphid, Aphis gossypii Glover (Hem., Aphididae), is one of the most important pests of cucumber throughout the world. This aphid has a short generation time and high fecundity that result in an enormous reproductive potential, especially in cucumber‐growing greenhouses. Vermicomposts, which are produced by exploiting interactions between earthworms and microorganisms, may enhance plant growth and plant resistance against some pests and disease. In this study, the effects of vermicompost and cucumber cultivar (Cucumis sativus L.) on infestation levels with A. gossypii were evaluated. We conducted a factorial experiment with two cucumber cultivars (Royal and Storm) and five concentrations of vermicompost in the soil, including 0% (control), 10%, 20%, 30% and 50%, employing a randomized complete block design with four replicates. The experiment was conducted in a growth chamber at 25 ± 2°C, 65 ± 10% RH and a photoperiod of 14 L: 10 D h. The number of aphids was counted 3, 5, 7, 9, 12, 15, 18 and 21 days after infestation of cucumber seedlings by aphids. We found that in all vermicompost‐amended treatments, aphid numbers were lower than when plants were grown in soil without any vermicompost. The highest and lowest aphid counts occurred in the control treatment on cucumbers of the Royal cultivar and in the 30% and 50% vermicompost treatments on the storm cultivar, respectively. Overall, our study showed that the application of vermicompost has a high potential for reducing A. gossypii populations in cucumber cultures.     

Host‐associated differentiation and evidence for sexual reproduction in Iranian populations of the cotton aphid,Aphis gossypii

Phytophagous insects with wide host ranges often exhibit host‐associated genetic structure. We used microsatellite analysis to assess the population structure of the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), a serious pest on many economically important crops worldwide. We sampled aphids from five host plant species in Iran and detected strong population subdivision, with an overall multilocus F_ST of 0.191. The matrix of pairwise F_ST values indicated that differentiation between populations collected from different hosts was significantly stronger than between populations from the same hosts. Host‐associated differentiation was further supported by Bayesian clustering analyses, which grouped all samples from cotton together with aubergine, and all samples from cucumber together with pumpkin and hibiscus. This adds to the growing body of evidence that many seemingly generalist aphids are in fact an assemblage of host‐specialized lineages. Although we detected a clear genetic signature of clonal reproduction, the genotypic diversity of A. gossypii in Iran is much higher than in other parts of the world. Particularly samples from cotton exhibited a surprisingly high genotypic diversity, suggesting that many lineages on this host are cyclical parthenogens that engage in regular bouts of sexual reproduction.     

Effects of elevated CO2 on the interspecific competition between two sympatric species of Aphis gossypii and Bemisia tabaci fed on transgenic Bt cotton

Abstract Effects of elevated CO₂ (twice ambient vs. ambient) and Bt Cry1Ac transgene (Bt cotton cv. 33^B vs. its nontransgenic parental line cv. DP5415) on the interspecific competition between two ecologically similar species of cotton aphid Aphis gossypii and whitefly biotype‐Q Bemisia tabaci were studied in open‐top chambers. The results indicated that elevated CO₂ and Bt cotton both affected the population abundances of A. gossypii and biotype‐Q B. tabaci when introduced solely (i.e., without interspecific competition) or two species coexisted (i.e., with interspecific competition). Compared with ambient CO₂, elevated CO₂ increased the population abundances of A. gossypii and biotype‐Q B. tabaci as fed on Bt and nontransgenic cotton on 45 (i.e., seedling stage) and 60 (i.e., flowering stage) days after planting (DAP), but only significantly enhanced aphid abundance without interspecific competition on the 45‐DAP nontransgenic cotton and 60‐DAP Bt cotton, and significantly increased whitefly abundance with interspecific competition on the 45‐DAP Bt cotton and 60‐DAP nontransgenic cotton. In addition, compared with nontransgenic cotton at elevated CO₂, Bt cotton significantly reduced biotype‐Q B. tabaci abundances without and with interspecific competition during seedling and flowering stage, while only significantly decreasing A. gossypii abundances without interspecific competition during the seedling stage. When the two insect species coexisted, the proportions of biotype‐Q B. tabaci were significantly higher than those of A. gossypii on Bt and nontransgenic cotton at the same CO₂ levels, and elevated CO₂ only significantly increased the percentages of biotype‐Q B. tabaci and significantly reduced the proportions of A. gossypii on seedling and flowering nontransgenic cotton. Therefore, the effects of elevated CO₂ were favorable for biotype‐Q B. tabaci to out‐compete A. gossypii under the predicted global climate change.     

Genetic diversity of the melon aphid <Emphasis Type="Italic">Aphis gossypii</Emphasis> (Hemiptera: Aphididae) in a diversified vegetable growing area

The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a highly polyphagous species. To investigate its genetic diversity among seasonal host plants, we conducted a field study in a diversified vegetable growing area in Beijing, China. The molecular marker mtDNA COI (1563-bp long) was used to analyze the genetic diversity of aphid populations collected from eight plant species belonging to families Malvaceae, Cucurbitaceae and Rosaceae. A total of 33 haplotypes were identified, five of which were shared haplotypes, while the remaining 28 were unique haplotypes. At least one haplotype was shared by all eight A. gossypii populations. Aphid populations showed high levels of nucleotide and haplotype diversity. The genetic diversity indices were maximal on the hibiscus (the primary host), whereas minimal on cucumber and strawberry (the secondary hosts). The analysis of molecular variance showed that most of the variance was distributed within populations. Based on the genetic distances, the eight populations can be divided into two groups, associated with primary and the secondary host plant, respectively. The aphids possibly migrated from Hibiscus to watermelon, thereafter dispersed from watermelon to other secondary host plants. Watermelon was an important host probably due to its early growing season and large planting area. Our results highlighted the need to target the population dispersal for effective control.     

Presence of two host races of Aphis gossypii Glover (Hemiptera: Aphididae) collected in Turkey

The aphid, Aphis gossypii, is a primary pest of citrus, cotton, cucurbits and greenhouse‐grown vegetables in Turkey and throughout Europe. There is some previous empirical data suggesting that host‐adapted genotypes of this aphid exist which may in fact be host‐races. To determine if host races of A. gossypii are indeed present in the eastern Mediterranean region of Turkey, reciprocal host transfer experiments and life table analyses were performed with multiple asexual lineages (= clones) of the aphid collected from different hosts. The collection hosts included citrus, cucumber, eggplant, okra, sweet pepper and cotton. Aphid developmental times on the host from which the aphid was originally collected (= collection or natal host) were shorter (5.2–6.0 days) and had a higher intrinsic rate of population growth (r_m = 0.25–0.44) than the 6.6–7.3 days required when the aphid was reared on a non‐original collection host (= non‐collection host or non‐natal host) and had r_m = 0.03–0.30. Total immature mortality of the cotton clone, especially in the first nymphal stage, was high (51–100%) with low r_m (0–0.03) on cucumber, citrus and sweet pepper. Aphid populations transferred from citrus, eggplant and okra to cotton (r_m = 0.29–0.30) did not differ significantly in their performance from that of the cotton population on cotton (r_m = 0.34), whereas that from sweet pepper and cucumber populations (r_m = 0.22–0.24) were significantly lower. These data have allowed us to separate A. gossypii into two distinct biological groups: (a) a ‘generalist’ population obtained from cucumber, sweet pepper, citrus, eggplant and okra which exhibited statistically better development on cotton; versus (b) a population from cotton which, by comparison on reciprocal hosts, developed poorly on non‐natal hosts except on eggplant. Development of the cotton clone on cucumber and okra was not improved after four successive generations on the non‐natal host. The good development of A. gossypii from eggplant and cotton on these reciprocal hosts suggests that these particular clones were similar, if not identical, host races.     

取食不同寄主植物对棉蚜后代抗药性的影响

测定了5种药剂对棉蚜Aphis gossypii抗氰戊菊酯、吡虫啉品系和敏感品系取食棉花、黄瓜和石榴的后代的毒力,并对它们的后代体内乙酰胆碱酯酶和羧酸酯酶的比活力做了初步探索。结果表明,氰戊菊酯抗性品系取食棉花比取食黄瓜的后代对氰戊菊酯的抗性大76.4倍,对灭多威、氧乐果、硫丹和吡虫啉的抗性也大0.5～4.6倍;取食石榴的后代对5种药剂的抗性介于取食棉花和黄瓜的之间。吡虫啉抗性品系的测定结果与氰戊菊酯抗性品系基本一致。敏感品系取食黄瓜比取食棉花的后代对5种药剂的敏感性更高。3个品系取食不同植物的后代相比,其体内乙酰胆碱酯酶的比活力,取食棉花的为取食黄瓜的2.4～2.8倍;羧酸酯酶的比活力,取食棉花的为取食黄瓜的1.8～2.4倍。证明棉蚜的抗性和敏感品系取食的寄主植物不同,可引起对药剂敏感性的变化。乙酰胆碱酯酶和羧酸酯酶活力的变化均是引起这种变化的重要因素。     

Contest and scramble competitions inCallosobruchus maculatus (Coleoptera: Bruchidae)

Larval competition curves and resource sharing patterns of 5 strains of Callosobruchus maculatus (iQ, yQ, aaQ, wQ, and tQ) were examined. Offspring emergences as a function of the initial larval density were recorded to construct competition curves. Elytron length of emerged adults was used as the indicator of resource sharing patterns among competing larvae inside a bean. In the large beans, strain iQ showed a saturated competition curve and tQ strain showed a humped curve. Competition curves of the other 3 strains (yQ, aaQ, and wQ) were between those two extremes. In the small beans, strains iQ and tQ also showed a saturated and a humped competition curves, respectively, whereas the competition curves of the 3 intermediate scramble strains could not be distinguished from that of the iQ strain. Thus, the classification based on competition curves was sensitive to the resource condition (bean size). In both the large and the small beans, the elytron lengths of iQ strain remained constant irrespective of initial larval density. On the contrary, the elytron lengths of the 4 other strains decreased monotonically with higher initial larval density. Thus, the judgment based on the resource sharing pattern was shown to be robust. Only iQ strain should be designated as a contest type, and the remaining strains as scaramble types. Contest and scramble types in C. maculatus were also compared with those observed in C. analis and C. phaseoli using competition curves, resource sharing patterns, and other physiological characters.     

Effect of PGPR on population growth parameters of cotton aphid

Plant-growth-promoting rhizobacteria (PGPR) help plants by increasing plant growth and defending them against pathogens and pests. There is considerable research supporting the use of these bacteria in plant pathogens management, but the number of research papers that have focused on their effect on insect pests control is quite few. In this research, we examined the ability of four native strains of Pseudomonas fluorescens to manage the cotton aphid, Aphis gossypii, on greenhouse-grown cucumbers in Iran. The strains UTPF (University of Tehran Pseudomonas fluorescens) 68, UTPF1, UTPF6 and PF169 of P. fluorescens were used as seed treatment to evaluate their impact on both cucumber growth and cotton aphid population growth parameters. The strains UTPF68 and PF169 of this bacterium influenced phenology of cucumber by decreasing the Growing Degree Days of flowering period. Also, the plants treated with these bacterial strains had a significant increase in yield weight of approximately 58%. In the other part of the study, various effects on developmental parameters of aphid were recorded among bacterial strains, but only PF169 caused significant decreasing effect on r_m value of aphid, which is the most important factor in aphid’s life table. We conclude that the PF169 treatment had significant negative effects on population growth rate of A. gossypii. The PF169 could be reliable in a control programme for A. gossypii on cucumber plants in greenhouses.     

棉花型和瓜型棉蚜产生有性世代能力的分化

为了明确寄主专化性是否会影响棉蚜的繁殖策略或生活史对策,采用低温和短光照组合（18℃,L∶D=8∶16）分别对棉花型和瓜型棉蚜进行有性世代的诱导,比较两寄主专化型棉蚜产生有翅蚜、雄性母、雌性母及无翅孤雌蚜的能力。结果表明：两寄主专化型棉蚜在产生有性世代能力上存在显著差异,表现为棉花型棉蚜在诱导后代中产生有翅蚜、雄性母和雌性母的比率显著高于瓜型棉蚜,并且雌、雄性母产生的时间明显早于瓜型棉蚜。瓜型棉蚜在诱导条件下产生无翅孤雌蚜的比率显著高于棉花型棉蚜,并且发现有不产生有性世代的营专性孤雌生殖个体,而在棉花型棉蚜中没有发现。产生性母蚜的棉花型和瓜型棉蚜均可同时产生孤雌胎生后代。寄主型与诱导时间长短对棉蚜有性世代的产生存在交互作用。采集于田间棉花和黄瓜上的棉蚜,也表现为棉花上的易产生有性世代,在诱导的第2代中产生性母蚜的比率显著高于黄瓜上的棉蚜,并且性母蚜比率在诱导的第2代与第3代间无显著差异; 但黄瓜上的棉蚜性母蚜产生比率第3代显著高于第2代。由此推测,棉蚜寄主专化性的形成与生活史特性的分化有关。     

An autoparasitoid wasp,inferior at resource exploitation,outcompetes primary parasitoids by using competitor females to produce males

1. Autoparasitoids are intraguild consumers that attack and kill heterospecific and conspecific parasitoids as well as immature stages of hemipteran hosts, such as aphids, whiteflies and soft scales. Field experiments assessing the importance of interspecific competition between autoparasitoids and primary parasitoids, as well as its impact on herbivore suppression, are scarcely found in the ecological literature. 2. Using field data from 40 olive orchards, this study examined the mechanisms that regulate: (i) the interspecific competition between primary parasitoids of the genus Metaphycus and the autoparasitoid Coccophagus lycimnia; and (ii) the density of their shared herbivore host, the soft scale Saissetia oleae. 3. Metaphycus parasitoids used smaller hosts than C. lycimnia, yet did not outcompete C. lycimnia. On the other hand, C. lycimnia preferred to use Metaphycus females as secondary hosts for producing males rather than their own females. This preference might explain why the autoparasitoid negatively affected the density of the primary parasitoids. 4. Parasitism by the autoparasitoid C. lycimnia at the beginning of the season was the sole variable positively related to host mortality throughout the season, showing its greater effect on herbivore suppression. 5. In this study, an autoparasitoid, inferior at resource exploitation, was shown to outcompete a primary parasitoid without disrupting herbivore suppression.     

The Entomopathogenic Fungal Endophytes Purpureocillium lilacinum (Formerly Paecilomyces lilacinus) and Beauveria bassiana Negatively Affect Cotton Aphid Reproduction under Both Greenhouse and Field Conditions

The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum (formerly Paecilomyces lilacinus), were assessed on the reproduction of cotton aphid, Aphis gossypii Glover (Homoptera:Aphididae), through in planta feeding trials. In replicate greenhouse and field trials, cotton plants (Gossypium hirsutum) were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia. Positive colonization of cotton by the endophytes was confirmed through potato dextrose agar (PDA) media plating and PCR analysis. Inoculation and colonization of cotton by either B. bassiana or P. lilacinum negatively affected aphid reproduction over periods of seven and 14 days in a series of greenhouse trials. Field trials were conducted in the summers of 2012 and 2013 in which cotton plants inoculated as seed treatments with B. bassiana and P. lilacinum were exposed to cotton aphids for 14 days. There was a significant overall effect of endophyte treatment on the number of cotton aphids per plant. Plants inoculated with B. bassiana had significantly lower numbers of aphids across both years. The number of aphids on plants inoculated with P. lilacinum exhibited a similar, but non-significant, reduction in numbers relative to control plants. We also tested the pathogenicity of both P. lilacinum and B. bassiana strains used in the experiments against cotton aphids in a survival experiment where 60% and 57% of treated aphids, respectively, died from infection over seven days versus 10% mortality among control insects. Our results demonstrate (i) the successful establishment of P. lilacinum and B. bassiana as endophytes in cotton via seed inoculation, (ii) subsequent negative effects of the presence of both target endophytes on cotton aphid reproduction using whole plant assays, and (iii) that the P. lilacinum strain used is both endophytic and pathogenic to cotton aphids. Our results illustrate the potential of using these endophytes for the biological control of aphids and other herbivores under greenhouse and field conditions.     

Do past experience and competitive ability influence foraging strategies of parasitoids under interspecific competition?

Abstract 1. In solitary parasitoids, several species can exploit the same host patch and competition could potentially be a strong selective agent as only one individual can emerge from a host. In cereal crops, Aphidius rhopalosiphi and A. ervi share the grain aphid Sitobion avenae as host. 2. The present work studied foraging strategies of both species on patches already exploited by the other species. The study analysed larval competition in multi‐parasitised hosts and compared the foraging behaviour of females with and without previous experience. 3. It was found that A. ervi wins larval competition three times more often than A. rhopalosiphi. Both species spent less time on patches exploited by a heterospecific than on unexploited ones. When they foraged on heterospecifically exploited patches, experienced females induced less mortality in aphids than inexperienced ones. 4. Although A. rhopalosiphi is a specialist on cereal aphids and is the most abundant species due to its early appearance in the season, S. avenae is still a profitable host for A. ervi, because: (i) A. rhopalosiphi leaves patches partially exploited, (ii) A. ervi wins larval competition in three out of four multi‐parasitised hosts, and (iii) A. ervi is only slightly deterred by the cornicular secretions of the host and can thus easily parasitise hosts.     

Invasive annual grasses indirectly increase virus incidence in California native perennial bunchgrasses

In California valley grasslands, Avena fatua L. and other exotic annual grasses have largely displaced native perennial bunchgrasses such as Elymus glaucus Buckley and Nassella pulchra (A. Hitchc.) Barkworth. The invasion success and continued dominance of the exotics has been generally attributed to changes in disturbance regimes and the outcome of direct competition between species. Here, we report that exotic grasses can also indirectly increase disease incidence in nearby native grasses. We found that the presence of A. fatua more than doubled incidence of infection by barley and cereal yellow dwarf viruses (B/CYDVs) in E. glaucus. Because B/CYDV infection can stunt E. glaucus and other native bunchgrasses, the indirect effects of A. fatua on virus incidence in natives suggests that apparent competition may be an additional mechanism influencing interactions among exotic and native grasses in California. A. fatua's influence on virus incidence is likely mediated by its effects on populations of aphids that vector B/CYDVs. In our study, aphids consistently preferred exotic annuals as hosts and experienced higher fecundity on them, suggesting that the exotics can attract and amplify vector populations. To the best of our knowledge, these findings are the first demonstration that exotic plants can indirectly influence virus incidence in natives. We suggest that invasion success may be influenced by the capacity of exotic plant species to increase the pathogen loads of native species with which they compete.     

Within-host competition between Borrelia afzelii ospC strains in wild hosts as revealed by massively parallel amplicon sequencing

Infections frequently consist of more than one strain of a given pathogen. Experiments have shown that co-infecting strains often compete, so that the infection intensity of each strain in mixed infections is lower than in single strain infections. Such within-host competition can have important epidemiological and evolutionary consequences. However, the extent of competition has rarely been investigated in wild, naturally infected hosts, where there is noise in the form of varying inoculation doses, asynchronous infections and host heterogeneity, which can potentially alleviate or eliminate competition. Here, we investigated the extent of competition between Borrelia afzelii strains (as determined by ospC genotype) in three host species sampled in the wild. For this purpose, we developed a protocol for 454 amplicon sequencing of ospC, which allows both detection and quantification of each individual strain in an infection. Each host individual was infected with one to six ospC strains. The infection intensity of each strain was lower in mixed infections than in single ones, showing that there was competition. Rank-abundance plots revealed that there was typically one dominant strain, but that the evenness of the relative infection intensity of the different strains in an infection increased with the multiplicity of infection. We conclude that within-host competition can play an important role under natural conditions despite many potential sources of noise, and that quantification by next-generation amplicon sequencing offers new possibilities to dissect within-host interactions in naturally infected hosts.