Ancestral haplotype reconstruction in endogamous populations using identity-by-descent.

In this work we develop a novel algorithm for reconstructing the genomes of ancestral individuals, given genotype or sequence data from contemporary individuals and an extended pedigree of family relationships. A pedigree with complete genomes for every individual enables the study of allele frequency dynamics and haplotype diversity across generations, including deviations from neutrality such as transmission distortion. When studying heritable diseases, ancestral haplotypes can be used to augment genome-wide association studies and track disease inheritance patterns. The building blocks of our reconstruction algorithm are segments of Identity-By-Descent (IBD) shared between two or more genotyped individuals. The method alternates between identifying a source for each IBD segment and assembling IBD segments placed within each ancestral individual. Unlike previous approaches, our method is able to accommodate complex pedigree structures with hundreds of individuals genotyped at millions of SNPs. We apply our method to an Old Order Amish pedigree from Lancaster, Pennsylvania, whose founders came to North America from Europe during the early 18th century. The pedigree includes 1338 individuals from the past 12 generations, 394 with genotype data. The motivation for reconstruction is to understand the genetic basis of diseases segregating in the family through tracking haplotype transmission over time. Using our algorithm thread, we are able to reconstruct an average of 224 ancestral individuals per chromosome. For these ancestral individuals, on average we reconstruct 79% of their haplotypes. We also identify a region on chromosome 16 that is difficult to reconstruct-we find that this region harbors a short Amish-specific copy number variation and the gene HYDIN. thread was developed for endogamous populations, but can be applied to any extensive pedigree with the recent generations genotyped. We anticipate that this type of practical ancestral reconstruction will become more common and necessary to understand rare and complex heritable diseases in extended families.

A population genetic interpretation of GWAS findings for human quantitative traits.

Human genome-wide association studies (GWASs) are revealing the genetic architecture of anthropomorphic and biomedical traits, i.e., the frequencies and effect sizes of variants that contribute to heritable variation in a trait. To interpret these findings, we need to understand how genetic architecture is shaped by basic population genetics processes-notably, by mutation, natural selection, and genetic drift. Because many quantitative traits are subject to stabilizing selection and because genetic variation that affects one trait often affects many others, we model the genetic architecture of a focal trait that arises under stabilizing selection in a multidimensional trait space. We solve the model for the phenotypic distribution and allelic dynamics at steady state and derive robust, closed-form solutions for summary statistics of the genetic architecture. Our results provide a simple interpretation for missing heritability and why it varies among traits. They predict that the distribution of variances contributed by loci identified in GWASs is well approximated by a simple functional form that depends on a single parameter: the expected contribution to genetic variance of a strongly selected site affecting the trait. We test this prediction against the results of GWASs for height and body mass index (BMI) and find that it fits the data well, allowing us to make inferences about the degree of pleiotropy and mutational target size for these traits. Our findings help to explain why the GWAS for height explains more of the heritable variance than the similarly sized GWAS for BMI and to predict the increase in explained heritability with study sample size. Considering the demographic history of European populations, in which these GWASs were performed, we further find that most of the associations they identified likely involve mutations that arose shortly before or during the Out-of-Africa bottleneck at sites with selection coefficients around s = 10-3.

Correction: The population genetics of human disease: The case of recessive, lethal mutations.

[This corrects the article DOI: 10.1371/journal.pgen.1006915.].

The population genetics of human disease: The case of recessive, lethal mutations.

Do the frequencies of disease mutations in human populations reflect a simple balance between mutation and purifying selection? What other factors shape the prevalence of disease mutations? To begin to answer these questions, we focused on one of the simplest cases: recessive mutations that alone cause lethal diseases or complete sterility. To this end, we generated a hand-curated set of 417 Mendelian mutations in 32 genes reported to cause a recessive, lethal Mendelian disease. We then considered analytic models of mutation-selection balance in infinite and finite populations of constant sizes and simulations of purifying selection in a more realistic demographic setting, and tested how well these models fit allele frequencies estimated from 33,370 individuals of European ancestry. In doing so, we distinguished between CpG transitions, which occur at a substantially elevated rate, and three other mutation types. Intriguingly, the observed frequency for CpG transitions is slightly higher than expectation but close, whereas the frequencies observed for the three other mutation types are an order of magnitude higher than expected, with a bigger deviation from expectation seen for less mutable types. This discrepancy is even larger when subtle fitness effects in heterozygotes or lethal compound heterozygotes are taken into account. In principle, higher than expected frequencies of disease mutations could be due to widespread errors in reporting causal variants, compensation by other mutations, or balancing selection. It is unclear why these factors would have a greater impact on disease mutations that occur at lower rates, however. We argue instead that the unexpectedly high frequency of disease mutations and the relationship to the mutation rate likely reflect an ascertainment bias: of all the mutations that cause recessive lethal diseases, those that by chance have reached higher frequencies are more likely to have been identified and thus to have been included in this study. Beyond the specific application, this study highlights the parameters likely to be important in shaping the frequencies of Mendelian disease alleles.

Symbiotic bacteria enable olive flies (Bactrocera oleae) to exploit intractable sources of nitrogen.

Insects are often associated with symbiotic micro-organisms, which allow them to utilize nutritionally marginal diets. Adult fruit flies (Diptera: Tephritidae) associate with extracellular bacteria (Enterobacteriaceae) that inhabit their digestive tract. These flies obtain nutrients by foraging for plant exudates, honeydew and bird droppings scattered on leaves and fruit­a nutritional niche which offers ample amounts of carbohydrates, but low quantities of available nitrogen. We identified the bacteria resident in the gut of the olive fly (Bactrocera oleae)­a worldwide pest of olives and examined their contribution to nitrogen metabolism in the adult insect. By suppressing bacteria in the gut and monitoring female fecundity, we demonstrate that bacteria contribute essential amino acids and metabolize urea into an available nitrogen source for the fly, thus significantly elevating egg production. In an ecological context, bacteria were found to be beneficial to females subsisting on bird droppings, but not on honeydew­two natural food sources. We suggest that a main gut bacterium (Candidatus Erwinia dacicola) forms an inseparable, essential part of this fly's nutritional ecology. The evolution of this symbiosis has allowed adult flies to utilize food substrates which are low or imbalanced in assimilable nitrogen and thereby to overcome the nitrogen limitations of their natural diet.

Conditional frequency spectra as a tool for studying selection on complex traits in biobanks.

Natural selection on complex traits is difficult to study in part due to the ascertainment inherent to genome-wide association studies (GWAS). The power to detect a trait-associated variant in GWAS is a function of frequency and effect size - but for traits under selection, the effect size of a variant determines the strength of selection against it, constraining its frequency. To account for GWAS ascertainment, we propose studying the joint distribution of allele frequencies across populations, conditional on the frequencies in the GWAS cohort. Before considering these conditional frequency spectra, we first characterized the impact of selection and non-equilibrium demography on allele frequency dynamics forwards and backwards in time. We then used these results to understand conditional frequency spectra under realistic human demography. Finally, we investigated empirical conditional frequency spectra for GWAS variants associated with 106 complex traits, finding compelling evidence for either stabilizing or purifying selection. Our results provide insight into polygenic score portability and other properties of variants ascertained with GWAS, highlighting the utility of conditional frequency spectra.

Diel rhythm of volatile emissions of males and females of the peach fruit fly Bactrocera zonata.

Fruit flies in the genus Bactrocera are among the most destructive insect pests of fruits and vegetables throughout the world. A number of studies have identified volatiles from fruit flies, but few reports have demonstrated behavioral effects or sensitivities of fly antennae to these compounds. We applied a recently developed method of automated headspace analysis using SPME (Solid Phase Microextraction) fibers and GC-MS (gas chromatography mass spectrometry), termed SSGA, to reveal volatiles specific to each sex of B. zonata that are emitted in a diel periodicity. The volatiles released primarily at dusk were identified by GC-MS and chemical syntheses as several spiroacetals, pyrazines, and ethyl esters. Solvent extraction of male rectal glands or airborne collections from each sex, followed by GC-MS, showed that certain of the volatiles increase or decrease in quantity sex-specifically with age of the flies. Electroantennographic (EAG) analysis of dose-response indicates differences in sensitivities of male and female antenna to the various volatiles. Our study provides a comprehensive analysis of the volatile chemicals produced and released by B. zonata and their antennal responses. The possible pheromone and semiochemical roles of the various volatiles released by each sex and the difficulties of establishing behavioral functions are discussed.

Bringing back the fruit into fruit fly-bacteria interactions.

Female Mediterranean fruit flies (Ceratitis capitata) oviposit in fruits, within which the larvae develop. This development is associated with rapid deterioration of the fruit, and frequently with invasion by secondary pests. Most research on the associations between medflies and microorganisms has focused on the bacteria inhabiting the digestive system of the adult fly, while the role of the fruit in mediating, amplifying or regulating the fruit fly microflora has been largely neglected. In this study, we examine the hypothesis that the host fruit plays a role in perpetuating the fly-associated bacterial community. Using direct and cultured-based approaches, we show that this community is composed in its very large majority of diazotrophic and pectinolytic Enterobacteriaceae. Our data suggest that this fly-associated enterobacterial community is vertically transmitted from the female parent to its offspring. During oviposition, bacteria are transferred to the fruit, establish and proliferate within it, causing its decay. These results show that the host fruit is indeed a central partner in the fruit fly-bacterial interaction as these transmitted bacteria are amplified by the fruit, and subsequently maintained throughout the fly's life. This enterobacterial community may contribute to the fly's nitrogen and carbon metabolism, affecting its development and ultimately, fitness.

Gut bacterial communities in the Mediterranean fruit fly (Ceratitis capitata) and their impact on host longevity.

Fruit flies (Diptera: Tephritidae) harbor stable bacterial communities in their digestive system, composed mainly of members of the Enterobacteriaceae. However, the Enterobacteriaceae are not the sole community in this habitat. We examined the hypothesis that Pseudomonas spp. form a cryptic community in the gut of Ceratitis capitata, the Mediterranean fruit fly ('medfly'). Suicide polymerase restriction PCR (SuPER PCR), a novel culture-independent technique, revealed that Pseudomonas spp. form minor, common and stable communities within the medfly's gut. These include P. aeruginosa, a known pathogen of arthropods. Experimental inoculations with high levels of P. aeruginosa reduced the medfly's longevity while inoculations with members of the Enterobacteriaceae extended the fly's life. Accordingly, we suggest that in addition to their possible contribution to the fly's nitrogen and carbon metabolism, development and copulatory success (as shown in previous studies), the Enterobacteriaceae community within the medfly's gut may also have an indirect contribution to host fitness by preventing the establishment or proliferation of pathogenic bacteria.

Verrucous carcinoma of the scalp.

Verrucous carcinoma is a distinctive form of low-grade squamous cell carcinoma. It typically involves the oral cavity, larynx, esophagus, and skin. Cutaneous lesions typically arise in the genitocrural area and plantar surface of the foot, with rare case reports of verrucous carcinoma arising elsewhere on the body. Human papillomaviruses, predominately types 6 and 11, have been associated with some case reports. We present a case of verrucous carcinoma arising on the scalp with negative human papillomavirus testing in a relatively young patient.

The impact of recent population history on the deleterious mutation load in humans and close evolutionary relatives.

Over the past decade, there has been both great interest and confusion about whether recent demographic events-notably the Out-of-Africa-bottleneck and recent population growth-have led to differences in mutation load among human populations. The confusion can be traced to the use of different summary statistics to measure load, which lead to apparently conflicting results. We argue, however, that when statistics more directly related to load are used, the results of different studies and data sets consistently reveal little or no difference in the load of non-synonymous mutations among human populations. Theory helps to understand why no such differences are seen, as well as to predict in what settings they are to be expected. In particular, as predicted by modeling, there is evidence for changes in the load of recessive loss of function mutations in founder and inbred human populations. Also as predicted, eastern subspecies of gorilla, Neanderthals and Denisovans, who are thought to have undergone reductions in population sizes that exceed the human Out-of-Africa bottleneck in duration and severity, show evidence for increased load of non-synonymous mutations (relative to western subspecies of gorillas and modern humans, respectively). A coherent picture is thus starting to emerge about the effects of demographic history on the mutation load in populations of humans and close evolutionary relatives.

Clioquinol effects on tissue chelatable zinc in mice.

Recent evidence for the involvement of zinc in the formation of beta-amyloid plaques in the brain in Alzheimer's disease has led to the establishment of new therapeutic strategies for the degenerative disorder based on metal chelation. The present experiment was conducted on a membrane-permeable zinc chelator, clioquinol (CQ), that has shown potential in initial studies on a mouse model of Alzheimer's disease [1]. The degree of chelatable zinc in mice treated with CQ, delivered by two different routes, was measured using complementary protocols for identifying chelatable zinc: 6-methoxy-8-quinolyl- p-toluenesulfonamide (TSQ) histofluorescence, and selenite autometalography. Mice injected intraperitoneally with CQ showed a dramatic reduction in chelatable zinc in brain, testis, and pancreas. In contrast, mice given CQ orally showed no significant change in levels of chelatable zinc in these tissues. This suggests that CQ administered orally to patients with Alzheimer's disease should not significantly perturb chelatable zinc levels in key organs and may be used over long periods without adverse endocrinological and reproductive effects related to zinc deficiency. In contrast, CQ injected intraperitoneally may be used not only as a tool for investigating chelatable zinc pools but also in a clinical context. For example, injected CQ could be employed in situations requiring the rapid buffering of excessive chelatable zinc following ischemic episodes or brain trauma. Thus, our findings indicate that CQ has considerable potential as a versatile scientific and clinical tool used for selective modulation of zinc pools.

Histochemical and histofluorescence tracing of chelatable zinc in the developing mouse.

Zinc is an essential element in mammalian development. However, little is known about concentrations of zinc in specific regions/organs in the embryo. We have employed selenite autometallography (AMG) and TSQ histofluoroscence to detect histochemically reactive (chelatable) zinc in whole midsagittal embryos and sections from neonatal mice. Chelatable zinc exhibited a broad distribution, being particularly localized to rapidly proliferating tissues, such as skin and gastrointestinal epithelium. Zinc was also observed in various types of tissues such as bone and liver. In the perinatal central nervous system, zinc was present almost exclusively in choroid plexus. The two methods used demonstrated generally similar distributions with some exceptions, e.g., in liver and blood. The ubiquity of zinc in the embryo, particularly in rapidly proliferating tissues, suggests a widespread role in fetal physiology.

Multiple blood feeding in Anopheles freeborni (Diptera: Culicidae).

The frequency of multiple blood feeding in field populations of Anopheles freeborni was determined. Fifty-six laboratory-reared mosquitoes, all known to have taken two blood meals, were used to define the limits of the histologic procedure we used. Seventy-eight percent of known double meals were detected when the interval between meals was from 1 to 24 hr, and the time from the second meal until fixation ranged from 0 to 24 hr. At intervals outside this range, 50% of multiple meals were detected. In field-collected An. freeborni, the most important histologic parameters for determination of multiple feeding were the amount of heme that developed around each meal, the peritrophic membrane, partially digested blood meals, and physical blood meal separation. Examination of 134 blood-engorged An. freeborni collected in the field showed that 9.7% had imbibed multiple blood meals. These results suggest that multiple blood meals in An. freeborni are more frequent than previously thought, perhaps with significant epidemiologic implications.

Postnatal regulation of ZnT-1 expression in the mouse brain.

We have characterized the postnatal development of ZnT-1, a putative zinc transporter, in the mouse brain with respect to chelatable zinc in four distinct brain areas: cerebral cortex, hippocampus, olfactory bulb and cerebellum. At birth, both zinc and ZnT-1 immunoreactivity were nearly undetectable. Beginning at the end of the first postnatal week, ZnT-1 expression increased significantly in all areas examined except the cerebellum, which contains virtually no synaptic zinc. Moreover, neurons immunoreactive for ZnT-1 were typically present in areas rich in synaptic zinc, which increased in parallel with ZnT-1. In the cerebellum, in contrast, Purkinje cells exhibited robust immunoreactivity for ZnT-1 only in the second postnatal week. While the parallel development of zinc and ZnT-1 in forebrain regions supports a direct role for synaptic zinc in regulating ZnT-1 expression, ZnT-1 in cerebellar Purkinje cells could indicate that expression of this zinc transporter may also be regulated by a non-synaptic pool of zinc or by other mechanism(s). The striking developmental regulation of ZnT-1 expression together with synaptic zinc indicates that ZnT-1 may play a key role in protecting developing neurons against potentially toxic zinc.

Temporal dynamics of mating and predation in mosquito swarms.

We determined the numbers of copulations and predatory attacks in swarms ofAnopheles freeborni (Diptera: Culicidae), and the distribution of these events throughout the duration of the swarming period each day. On 19 evenings of observation, we recorded 2724 copulating pairs leaving swarms and 1351 dragonfly (Pantala hymenaea andErythemis collocata) attacks. Mating activity partially coincided with predator activity. Most copulations occurred between 10 and 20 min after the swarms formed, while predation events were most frequent during the initial 15 min of the swarm. We calculated the ratio of copulations to predatory attacks during the swarming period. This ratio was significantly higher in an area sheltered by trees than it was in the open. We suggest that physiological and ecological constraints other than predation operate on the mating system of this anopheline to affect the timing of swarm initiation and swarm site selection.

Effects of energetic reserves on behavioral patterns of Mediterranean fruit flies (Diptera: Tephritidae).

The objective of this study was to associate levels of nutritional reserves (specifically lipids, sugars, and glycogen) in individual Mediterranean fruit flies, Ceratitis capitata (Diptera: Tephritidae), with observed patterns of behavior in the field. We collected females (n=255) and males (n=181) from the field, recording the time of collection and the activity they were engaged in when collected. Subsequently, we employed colorimetric biochemical techniques to determine the precise amounts of lipids, sugars, and glycogen in each individual. Lipid and sugar levels in males varied significantly according to the time of collection and the type of activity. Lipid and sugar levels in females did not vary in this manner. Sugar levels in both males and females were highest during the evening, when most feeding occurs. Males that engaged in sexual signaling in leks during the mid-afternoon had relatively low sugar and high lipid levels. Males engaged in the alternative mating tactic of fruit guarding had relatively high sugar and low lipid contents. Glycogen levels in males were high in the mornings, and a decline in glycogen content was associated with participation in leks; however, female glycogen levels did not vary significantly with time of day or activity. Our results provide quantitative evidence for the role nutrient reserves play in driving patterns of male reproductive behavior, yet suggest that factors other than sugar and lipid reserves constrain female behavior.

Populations of Phlebotomus papatasi (Diptera: Psychodidae) and the risk of Leishmania major transmission in three Jordan Valley habitats.

The abundance, population structure, and Leishmania infection rates of Phlebotomus papatasi were studied at two villages, a 10-yr old date plantation, and an undisturbed natural habitat in the Jordan Valley throughout one season. On 109 trap nights in the villages, 53 female and 61 male P. papatasi were caught, whereas in burrows in the natural and agriculturally modified habitat, greater than 3,500 sandflies were trapped on 157 trap nights. Burrows in the data plantation produced larger numbers of males and females significantly more frequently than burrows in the natural habitat. The sex ratio in the natural habitat and the date plantation was significantly male biased. Parous rates were similar among habitats, whereas gravid females were significantly more active in the modified habitat. Leishmania infected females were not collected in the villages, yet close to 10% of females collected in the two other habitats were infected. Risk of transmission to humans in the villages was negligible, but people working in the date plantation were exposed to Leishmania infected sandflies.

Duration and regulation of the developmental cycle of Ixodes dammini (Acari: Ixodidae).

To determine how long the various developmental stages of the deer tick (Ixodes dammini Spielman, Clifford, Piesman & Corwin) survive in nature and to establish the interval between blood feeding and ecdysis or oviposition as well as subsequent larval eclosion, we observed ticks confined in the field. Unless adults feed during their first season (fall through spring) of activity, they die and do not survive the summer. Nonfed nymphs survive through two seasons (May through August) of feeding activity such that annual cohorts overlap. Nonfed larvae survive less than one year, and because they hatch toward the end of the summer, cohorts of this developmental stage do not overlap. Larvae that feed before September molt promptly and overwinter as nymphs; those that feed later overwinter engorged and ecdyse during the following spring. Fed nymphs fail to survive the winter, and thus must feed before late summer. They develop to the adult stage in the same year in which they feed. Regardless of time of feeding, females lay eggs in early summer, and the resulting larvae synchronously eclose late in that season. Although the cycle of this tick can be completed in nature in two years, it may extend to four years when hosts are relatively scarce. The seasonal inversion of larvae and nymphs appears to be regulated by physiological mechanisms and by host abundance.

Spatial distribution of adult mosquitoes (Diptera:Culicidae) in habitats associated with the rice agroecosystem of northern California.

The objective of this study was to determine whether abundance, blood feeding rates, and sex ratios of adult Anopheles freeborni Aitken and Culex tarsalis Coquillett were associated significantly with either rice field, pasture, riparian, or mixed habitats found within the rice culture agroecosystem of northern California. Significantly higher numbers of adult An. freeborni occurred in riparian and mixed habitats compared with rice and pasture habitats. Such a pattern was not evident for Cx. tarsalis. Riparian and pasture habitats contained significantly higher proportions of blood fed An. freeborni females than did rice and mixed habitats; however, the proportions of blood fed Cx. tarsalis females did not vary significantly among habitat types. The proportions of blood fed An. freeborni and Cx. tarsalis females in riparian habitats decreased with increasing abundance. There was no correlation between blood feeding rates and abundance for An. freeborni and Cx. tarsalis females in the other habitat types. The sex ratio of An. freeborni in pasture and riparian habitats was significantly female biased, unlike the other habitats which did not differ significantly from unity (1:1). Overall, riparian and mixed habitats contained greater numbers of adults mosquitoes; therefore, surveillance and control efforts of these mosquito species should be focused on such habitats.