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Learning Objectives

  • Define natural selection

Darwin and Descent with Modification

Charles Darwin is best known for his discovery of natural selection. In the mid-nineteenth century, the actual mechanism for evolution was independently conceived of and described by two naturalists: Charles Darwin and Alfred Russel Wallace. Importantly, each naturalist spent time exploring the natural world on expeditions to the tropics. From 1831 to 1836, Darwin traveled around the world on H.M.S. Beagle, including stops in South America, Australia, and the southern tip of Africa. Wallace traveled to Brazil to collect insects in the Amazon rainforest from 1848 to 1852 and to the Malay Archipelago from 1854 to 1862. Darwin’s journey, like Wallace’s later journeys to the Malay Archipelago, included stops at several island chains, the last being the Galápagos Islands west of Ecuador. On these islands, Darwin observed species of organisms on different islands that were clearly similar, yet had distinct differences. For example, the ground finches inhabiting the Galápagos Islands comprised several species with a unique beak shape (Figure 1).

The species on the islands had a graded series of beak sizes and shapes with very small differences between the most similar. He observed that these finches closely resembled another finch species on the mainland of South America. Darwin imagined that the island species might be species modified from one of the original mainland species. Upon further study, he realized that the varied beaks of each finch helped the birds acquire a specific type of food. For example, seed-eating finches had stronger, thicker beaks for breaking seeds, and insect-eating finches had spear-like beaks for stabbing their prey.

Wallace and Darwin both observed similar patterns in other organisms and they independently developed the same explanation for how and why such changes could take place. Darwin called this mechanism natural selection. Natural selection, also known as “survival of the fittest,” is the more prolific reproduction of individuals with favorable traits that survive environmental change because of those traits; this leads to evolutionary change.

For example, a population of giant tortoises found in the Galapagos Archipelago was observed by Darwin to have longer necks than those that lived on other islands with dry lowlands. These tortoises were “selected” because they could reach more leaves and access more food than those with short necks. In times of drought when fewer leaves would be available, those that could reach more leaves had a better chance to eat and survive than those that couldn’t reach the food source. Consequently, long-necked tortoises would be more likely to be reproductively successful and pass the long-necked trait to their offspring. Over time, only long-necked tortoises would be present in the population.

Natural selection, Darwin argued, was an inevitable outcome of three principles that operated in nature. First, most characteristics of organisms are inherited, or passed from parent to offspring. Although no one, including Darwin and Wallace, knew how this happened at the time, it was a common understanding. Second, more offspring are produced than are able to survive, so resources for survival and reproduction are limited. The capacity for reproduction in all organisms outstrips the availability of resources to support their numbers. Thus, there is competition for those resources in each generation. Both Darwin and Wallace’s understanding of this principle came from reading an essay by the economist Thomas Malthus who discussed this principle in relation to human populations. Third, offspring vary among each other in regard to their characteristics and those variations are inherited. Darwin and Wallace reasoned that offspring with inherited characteristics which allow them to best compete for limited resources will survive and have more offspring than those individuals with variations that are less able to compete. Because characteristics are inherited, these traits will be better represented in the next generation. This will lead to change in populations over generations in a process that Darwin called descent with modification. Ultimately, natural selection leads to greater adaptation of the population to its local environment; it is the only mechanism known for adaptive evolution.

Papers by Darwin and Wallace (Figure 2) presenting the idea of natural selection were read together in 1858 before the Linnean Society in London. The following year Darwin’s book, On the Origin of Species, was published. His book outlined in considerable detail his arguments for gradual changes and adaptive survival by natural selection.

Demonstrations of evolution by natural selection are time consuming and difficult to obtain. One of the best examples has been demonstrated in the very birds that helped to inspire Darwin’s theory: the Galápagos finches. Peter and Rosemary Grant and their colleagues have studied Galápagos finch populations every year since 1976 and have provided important demonstrations of natural selection. The Grants found changes from one generation to the next in the distribution of beak shapes with the medium ground finch on the Galápagos island of Daphne Major. The birds have inherited variation in the bill shape with some birds having wide deep bills and others having thinner bills. During a period in which rainfall was higher than normal because of an El Niño, the large hard seeds that large-billed birds ate were reduced in number; however, there was an abundance of the small soft seeds which the small-billed birds ate. Therefore, survival and reproduction were much better in the following years for the small-billed birds. In the years following this El Niño, the Grants measured beak sizes in the population and found that the average bill size was smaller. Since bill size is an inherited trait, parents with smaller bills had more offspring and the size of bills had evolved to be smaller. As conditions improved in 1987 and larger seeds became more available, the trend toward smaller average bill size ceased.

How Could Language Have Evolved?

Affiliations Cognitive Neurobiology and Helmholtz Institute, Departments of Psychology and Biology, Utrecht University, Utrecht, The Netherlands, Department of Zoology and Sidney Sussex College, University of Cambridge, Cambridge, United Kingdom

Affiliation Division of Anthropology, American Museum of Natural History, New York, New York, United States of America

Affiliation Department of Linguistics and Philosophy, MIT, Cambridge, Massachusetts, United States of America

Affiliation Department of Electrical Engineering & Computer Science and Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, United States of America

Natural selection and mammalian BRCA1 sequences: elucidating functionally important sites relevant to breast cancer susceptibility in humans

Comparison of orthologous gene sequences is emerging as a powerful approach to elucidating functionally important positions in human disease genes. Using a diverse array of 132 mammalian BRCA1 (exon 11) sequences, we evaluated the functional significance of specific sites in the context of selection information (purifying, neutral, or diversifying) as well as the ability to extract such information from alignments that index varying degrees of mammalian diversity. Small data sets of either closely related taxa (Primates) or divergent placental taxa were unable to distinguish sites conserved due to purifying selection from sites conserved due to chance (false-positive rate = 65%-99%). Increasing the number of placental taxa to 57 greatly reduced the potential false-positive rate (0%-1.5%). Using the larger data set, we ranked the oncogenic risk of human missense mutations using a novel method that incorporates site-specific selection level and severity of the amino acid change evaluated against the amino acids present in other mammalian taxa. In addition to sites undergoing positive selection in Marsupialia, Laurasiatheria, Euarchontoglires, and Primates, we identified sites most likely to be undergoing divergent selection pressure in different lineages and six pairs of potentially interacting sites. Our results demonstrate the necessity of including large numbers of sequences to elucidate functionally important sites of a protein when using a comparative evolutionary approach.

2. Cat Flea Biology and Ecology

Several general reviews of C. f. felis biology have been published since 1997 [3,4,5,6,7,8,9,10,11]. Our understanding regarding the geographical distribution of C. f. felis and its alternate hosts continues to expand. C. f. felis is truly a global pest and global warming will probably not affect the distribution of cat fleas. The low outside persistence of C. f. felis, indoor breeding sites, a highly specialized life cycle, and a need for specific temperature and humidity conditions for development are all factors that suggest the distribution of cat fleas will remain the same [12]. However, with increased temperatures, the number of generations per year and potential density of cat fleas might dramatically increase.

Cat fleas belong to the Order Siphonaptera and the family Pulicidae. Within the family Pulicidae, the genus Ctenocephalides has undergone some major revisions with the advent of molecular systematics and critical reviews of existing morphological characters. Characters on the aedeagus such as the hamulus, lobes and tubus interior permit the identification of most of the species of Ctenocephalides [13]. However, the existence of morphological variations of characters used to differentiate C. f. felis and C. canis require that host data, geographical distribution, and the prevalence of infestations also be used in their determination [14,15]. From a systematic perspective, four subspecies of cat fleas had existed for six decades namely, C. felis damarensis, C. felis felis, C. felis orientis, and C. felis strongylus [16]. ITS1 and ITYS2 nucleotide sequences and 16SrDNA sequences were invariant in a number of C. felis populations collected worldwide and overall findings did not support subspecies of C. felis [17]. Several microsatellites have been identified that could help determine if host specific strains of C. f. felis exist, the existence of subspecies, and detailed epidemiological studies of Rickettsia felis [18]. Sequences of cytochrome c oxidase subunits cox1 and cox 2 indicate that C. f. felis and C. f. strongylus are paraphyletic and C. f. orientis is monophyletic [19]. Three distinct clades of C. f. felis were found. Similar studies with subunits cox1 and cox2 revealed that C. f. felis from New Zealand belonged to Clade 1 like those of Australia and Europe [20]. No intraspecific variation was found at the ITS1 marker for 52 C. f. felis specimens analyzed from 17 different locations in south central US, suggesting either a genetic bottleneck or that they were recently introduced [21]. Populations of C. f. felis and C. canis from Spain, Iran and South Africa were examined and ITS1 sequences conducted. Both species were clearly separated [22]. A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique was used to identify important pest species of fleas. A single fresh specimen provided unequivocal identification to species. Specimens preserved in ethanol provided variable results depending upon the length of time in ethanol [23].

Recent systematic efforts including molecular techniques have elevated two of the subspecies to full species, C. damarensis and C. orientis [13,24]. C. f. felis was found only on cats and dogs whereas C. f. strongylus was only found on large farm animals in Libya [25]. In South Africa, C. f. strongylus has been collected on the wild cat Caracal caracal and domestic dogs in rural areas [26]. Possibly C. f. strongylus will also be elevated to species status in the future.

For brevity C. felis felis will be referred to as C. felis.

2.1. Geographical Distribution and Hosts

Numerous surveys of the ectoparasites of companion animals have been conducted worldwide and they are briefly reviewed in order according to the continent, region and country. A review of the fleas of the hosts belonging to family Canidae indicate that C. felis is the most common flea of domesticated dogs globally [27]. C. felis has been collected on feral animals such as opossums, fox, rats, mongoose, and hedgehogs and this data are summarized in Table 1 . In general the numerous reports confirm that cats are more often infested by C. felis than dogs the prevalence of C. felis is seasonal, but it appears throughout the year and female fleas are collected more often than males. C. canis is more prevalent on dogs in some countries such as Greece, Iran, and Turkey.

Table 1

Summary of C. felis hosts other than cats and dogs a .

Species (Colloquial Name, Scientific name)Region(s)/CountriesCommentsKey References
African pygmy hedgehog Atelerix albiventrisTanzania2nd most prevalent ectoparasite[28]
Common opossum Didelphis masupialisFrench Guiana [29]
Domesticated ass Equus asinusIsraelsevere anaemia[30]
Domesticated sheep Ovis ariesIsrael, Iran, EthiopiaSeasonal allergic dermatitis[31,32,33]
Eastern cottontail rabbit Sylvilagus floridianusUnited Statesin zoo setting[34]
European hedgehog Erinaceus europaeusGermany7.9% hedgehogs infested[35]
Gazellas Gazelle gazelleIsraelin zoo[36]
Goat Capra aegagrus hircusEygpt, Iran, Ethiopia [32,33,37]
Golden cat Catopuma temminckiiThailand [38]
Gray fox Urocyon cinereoargenteusMexico [39]
Grizzly bear Ursus arctos horribilisUnited Statesin zoo[34]
Least weasel Mustela nivalisEygptserological study[37]
Maned wolves Chrysocyon brachyurusBrazilin zoo[40]
Margay Leopardus wiediiPeruin zoo[41]
Marsh deer Blastocercecus dichotomusBrazilin zoo[42]
Norway rat Rattus norvegicusChina, Eygpt [37,43]
Raccoon Procyon lotorWest Virginia, Virginia, United States [44,45]
Red fox Vulpes vulpesVirginia, South Carolina, United States [45,46]
Roof rat Rattus rattusEygpt [37]
Rüppel’s fox Vulpes rueppelliEygptserological study[37]
South American coati Nasua nasuaBrazilurban forests[47]
Striped skunk Mephitis mephitisConnecticut, United States [48]
Viriginia opossum Didelphis virginianaUnited States [34,45,46,49]
Water buffalo Bubalus bulalisIndia [50]

a Linardi and Santos [14] reported 41 species of mammals and 1 bird species in Brazil.

In Nigeria, 200 cats were examined of which 13% had C. felis [51]. In Hawassa, Ethiopia, there was a high incidence of ectoparasites on cats and dogs with 67% of cats and 82.9% of dogs infested with C. felis [52]. C. felis is not common in South Africa, but was taken from a dog in Johannesburg [26].

In a survey of 214 dogs, 110 (51.4%) were seropositive for anti-flea IgE in Japan indicating that dogs had been infested at one time. Dogs in northern areas of Japan thought to be flea free were also seropositive [53]. A survey of 324 stray dogs in India indicated that 24% were infested of which C. felis comprised 10.4% [54]. In Thailand, only C. felis were found on cats whereas C. orientis was found on dogs [55]. Stray cats (n = 200) in Taipei were examined and 82% were infested with C. felis [56]. A survey of 83 dogs from three regions of Iran resulted in 407 fleas of which 67.5% were C. felis [57]. In another study, along the Iraq-Iran border, 802 dogs and 50 cats were surveyed of which 2.4% of dogs and 65% of the cats were infested with C. felis [58]. Only 2 of 126 dogs in southwest Iran were infested with C. felis [59]. Of the fleas collected from 70 stray dogs in northern and central Iran, 19.9% of them were C. felis [60]. In a study in Israel, 340 stray cats were examined of which 54.7% were infested with C. felis. Fleas were recovered every month with highest numbers in the autumn [61].

In Australia, 98.8% of the 2500 fleas collected were C. felis and a single haplotype of the cox2 gene sequence was found [62]. In Borneo, 195 dogs were examined and 1965 fleas collected of which 25.4% were C. felis, the remaining being C. orientis [63]. C. felis was collected from both cats and dogs in Guam [64].

Many of the recent surveys have been conducted throughout Europe. A survey of ectoparasites and endoparasites of 1519 cats from seven countries in Europe found that 15.5% of the cats brought to veterinary clinics were infested with fleas. Of the cats suffering from anemia 93.5% were highly infested with fleas [65]. In southern Poland, of the 225 parasitic insects collected from pets only 3 were C. felis [66]. When dogs and cats were surveyed in the Czech Republic, 60% were C. felis, belonging to the cosmopolitan cox1 haplotype. A novel haplotype was found in both the Czech Republic and Romania [67]. A survey of 1342 dogs and 1378 cats presented to 22 different clinics in Serbia found that 79.2% of the fleas were C. felis with the most being found on cats from July to September [68]. In Hungary, 2267 dogs were inspected of which 115 dogs were infested with C. felis and 23 of 100 cats inspected were infested with C. felis. Fleas were more prevalent from rural animals than from urban animals [69]. In western Hungary, 71% of the 82 cats examined had C. felis [70]. In Turkey, 48 dogs were examined and 43.8% were infested with fleas. C. felis was found on 4.2% of the dogs with an average 5 fleas/dog. There were no seasonal patterns [71]. In Tirana, Albania, 128 dogs and 26 cats were examined for ectoparasites of which 5% of the dogs and 100% of the cats were infested with C. felis. Fleas were encountered year round [72]. In another study, from Tirana, 131 domestic cats were examined for ectoparasites with 52% being infested with C. felis. C. felis were collected year round with 48.8% being taken in the autumn from September to November [73]. Four clinics in Albania examined 602 client-owned dogs and found that 3.0% were infested with C. felis [74]. A survey in Germany found that 5.1% of dogs and 14.3% of cats were infested with fleas. Of the fleas collected, 81.1% were C. felis and there were no differences in urban vs. rural infestation rates [75]. Another survey in Germany found that 71.1% of dogs and 83.5% of cats were infested with C. felis. The increased prevalence over last decades may be due to temperature controlled housing [35]. In France, 392 dogs infested with fleas were examined and 86.6% of the fleas were C. felis. C. felis were found throughout France indoors and outdoors. Of the fleas collected from dogs living at elevations 𾐀 m only 11.2% were C. felis compared with 32.5% C. canis [76].

Thirty-one clinics in the UK were surveyed and a total of 2653 dogs and 1508 cats were examined of which 21.1% of the cats and 6.8% of the dogs were infested. C. felis was the most common flea with 98.9% on cats and 93.2% on dogs [77]. Of the 138 fleas collected in the UK in the autumn and winter, 96% were C. felis. The adult female cat fleas continued to mature oocytes throughout the fall and winter [78].

In Serbia, of the 1484 dogs brought to clinics from several cities 26.3% were infested with fleas of which 71.9% were C. felis. The highest infestation rates were from June to October [79]. In Greece, 13.7% of fleas collected from dogs were C. felis, the remaining being C. canis [80]. A survey in southern Italy found C. felis on 16.3% of the 1376 dogs examined [81]. Fleas were detected throughout the year with the greatest prevalence being between June and October. In another study in Italy, 80.3% of the fleas collected from 73 dogs and 44 cats were C. felis [82]. Of the 3032 fleas collected from 1084 dogs from 42 locations in Spain, 81.7% were C. felis. C. felis were most abundant in early summer and late autumn [83]. In another survey from Spain, 77 veterinary clinics collected 1938 fleas from 217 cats of which 98.4% were C. felis. There were lower infestation rates in warm summer months and overall flea abundance was positively associated with rainfall [84]. In Greece, 341 stray cats were examined and 24.3% were infested with C. felis. Cats with long hair (Ϥ cm long) had significantly more ectoparasites than did short haired cats with 42.3% of the ectoparasites being C. felis [85]. Of 242 stray dogs examined, 46.2% were infested with either C. felis or C. canis in Greece [86].

In North America, a survey of 200 feral cats from north central Florida found that 92.5% of them were infested with C. felis. The highest flea counts were in June and July (16.6�.3 fleas per cat) and the lowest from August to September (7.7 to 8.4 fleas per cat) [87]. C. felis was reported from dogs in South Carolina [46]. In Georgia, 2518 fleas were collected from dogs of which 61% were C. felis. Three female fleas were collected for each male. The vast majority of fleas were collected from August through October [88]. Of 673 free-roaming cats examined in the central US, 71.6% had fleas of which 97.2% were C. felis [89]. C. felis is common and widespread flea of pets in West Virginia and Virginia. Fleas were found in every month with June, September and October being the highest and April the lowest [44,45]. In Mexico, about 30% of the dogs (1803) and cats (517) examined were infested with fleas. Of the 4215 fleas collected, 81.1% were C. felis. There were no seasonal variations in the flea prevalence [45]. Similarly, of the 358 cats included in another study 53% were infested with fleas. Of the 2985 fleas collected, 89% were C. felis [90]. In Aguasclientes, Mexico, 863 dogs were examined and 38% of the 629 fleas collected were C. felis with the higher prevalence in spring and summer [91]. On the island of St. Kitts, 26% of 100 stray cats were infested with C. felis [92]. Fleas were the most common ectoparasite in homes in Costa Rica with 83% of dogs being infested with C. felis [93].

In South America, the most extensive surveys have been conducted in Brazil. Eighty-eight domestic dogs that lived outdoors were surveyed monthly for one year and only C. felis were collected. There was no significant correlation between temperature and infestation index and there was a negative association between infestation index and rainfall [94]. Paz et al. [94] concluded that seasonal differences in C. felis were likely due to climatic conditions in specific regions of Brazil. In a similar study, dogs from a farm in Brazil were surveyed for 1 year and two species of flea collected, C. felis and P. irritans. The number of cat fleas was significantly greater on long haired dogs than on short haired dogs [95]. Of 292 cats submitted to a spay/neuter program, 60% were infested with C. felis [96]. In rural northeastern Brazil, 18 of 29 dogs were infested with C. felis [97]. In two rural regions of Brazil, of the 328 dogs examined C. felis were found on 43.9 to 87.3% of the dogs depending upon the locality and season of the year [98]. In northeastern Brazil, 300 urban and 322 rural dogs were examined and 23.2% were infested with C. felis. More rural dogs were infested than urban dogs [99]. In Brazil, C. felis were the most common flea on dogs [100].

In other South American countries, 107 dogs from the domestic-wildlife interface in central Chile were surveyed and the following fleas were collected: C. felis (74.3%), C. canis (58.4%), Pulex sp. (11.8%) [101]. Studies in central Chile suggest that wild foxes (Pseudalopex griseus) and lesser grisons (Galictus cuja) could share fleas and potentially diseases with domesticated dogs. Fifty dogs were examined in Santiago, Concepción, and Osorno, Chile and of the 1000 fleas collected in each city C. felis represented 80.5, 38.4 and 6.6%, respectively. Osorno is more rural than the urban city of Santiago and this may explain the differences in species composition [102]. In Columbia, 140 dogs and 30 cats were surveyed and of the 3448 fleas collected 93.3% were C. felis [103]. C. felis was reported from French Guiana on cats and dogs [29].

C. felis is an opportunistic feeder and has been collected on wide range of feral hosts. A list of other hosts is provided in Table 1 .

2.2. Biology and Life History

Adult C. felis exhibited a circadian rhythm with maximum activity occurring about 9 h into the light phase [104]. Mating never occurred off host. Fleas must feed continuously for maximum mating and insemination to occur. Sperm transfer and the insemination of females by males fed on blood from membranes was 84 and 45%, respectively. The juvenile hormone analogues (JHs), methoprene and pyriproxyfen, may indirectly regulate mating success by stimulating sperm transfer [105]. Males fed salt solutions or protein free diets did not inseminate females [106]. The exposure of fleas to the host’s body temperature and the amount of feeding were two factors that influenced insemination [107]. The mating behavior of C. felis has been described in detail [108,109]. Only fed males attempted to mate with unfed or fed females. The majority of mating occurred at 38 ଌ which is the body temperatures of cats and dogs. Interestingly, a chloroform:methanol extract from virgin females appears to contain a sex pheromone. Female C. felis mated with many males [109].

About 25% of fleas collected from the pelage of cats were engorged within 5 min and nearly all had fed within 1 h. The average duration of feeding was 25 and 10 min for female and male fleas, respectively [110]. Female fleas produced significantly more dry fecal droplets than did males. However, male and female adult fleas produced similar amounts of protein in their fecal droplets [111].

Significantly more fleas were collected on the head and neck of cats compared with the ventral body. The fewest fleas were collected on legs and tail [56]. Once adult fleas have established themselves on a host (48 h), the movement to an uninfected host was low (3.7%) [112]. About 33% of the original fleas were unaccounted for after 72 h. Greater numbers of fleas were removed by grooming from flea allergic cats compared with normal cats. Female fleas produced fewer eggs on cats with flea allergic dermatitis (FAD) than did fleas on normal cats even though feeding was the same. Some unknown factor may have reduced the number of eggs produced on FAD cats [113]. The effectiveness of grooming by cats to remove cat fleas varied from 4.1 to 17.6% of its flea burden daily. The mean longevity of fleas on the host was 7.8 days and females laid 38.4 eggs per day [114]. Once fleas attain the host, host grooming appears to be a significant mortality factor.

A multiplexed PCR assay has been developed that can determine blood meals of fleas from humans, cats, chickens, and rats. Humans and cats were the main blood sources for C. f. strongylus [115]. Another advancement using real-time PCR was the ability to detect human, rat and goat DNA in C. felis artificially fed up to 72 h post feeding [116]. Another novel PCR technique was developed that is sensitive and specific for blood ingested by cat fleas [117]. Increased gene expression during blood feeding in C. felis was investigated and revealed a number of genes activated during feeding. The proteins from these genes may be important in blood digestion, cellular activities and protection during feeding. This may open new avenues for control [118]. The salivary constituents, sialome, of C. felis includes many small polypeptides of unknown function. Parts of the sialome of C. felis are similar to that of X. cheopis [119].

Aspects of jumping behavior have been investigated, suggesting that both C. canis and C. felis are highly adapted for securing large moving hosts. C. felis has a faster jumping speed (average 3.6 m/s) than X. cheopis (average 1.4 m/s) [120]. The mean height of jumps for C. canis was 15.5 cm and 13.2 cm for C. felis, the highest jump being 25 and 17 cm for C. canis and felis, respectively. The mean length of jumps for C. canis and felis was 30.4 and 19.9 cm, respectively [121].

When flea infested cats were kept in a carpeted room, the flea eggs and larvae accumulated around the pet’s feeding and resting places [122]. Linardi et al. [123] reported that blood from 9 different mammals and birds was inadequate nutrition with only 33% of larvae pupating. Early instars depend upon essential dietary components and consequently spend more time in those food patches. Larvae spent the most time in patches with adult fecal blood and flea eggs [124]. Spray-dried bovine blood was a satisfactory lab diet for cat flea larvae [125]. Only 13.3% of larvae developed in to adults when fed flea feces compared with 90% when fed flea feces and non-viable flea eggs. However, larvae did not develop on flea eggs alone [126]. All of the C. felis larvae that fed on adult fecal material and frozen cat flea eggs developed into adults whereas only 6.6% that fed on fecal blood developed into adults. Larvae consumed 㸠 flea eggs in developing into adults. This may serve as a population regulating factor [127]. There is a direct positive relationship between yeast consumption and cocoon formation [128]. Only 3rd instars ate eggs whereas all instars ate yeast. Naked pupae were consumed by 3rd instar larvae whereas pupae inside cocoons were protected from predation. Substrates such as carpet afford larvae protection from cannibalism and increased their chances to successfully develop in to adults. In addition to specific nutrients, the relative humidity in the environment is critical for development. Larvae actively uptake water when the RH > 53%. Pre-pupae actively uptake water when the RH is between 75 and 93%. Pupae and adults do not actively up take water from the atmosphere [129].

Larvae survived outdoors in north-central Florida year round. From September to November survival was as high as 84.6%. In June and July eggs developed into adults in 20� days whereas in the winter it took 36� days. Immature stages survived frosts in protected microhabitats [130]. Male prepupae and pupae develop about 20% slower than do females [130,131]. At 15.5 ଌ, some adults emerge as late as 155 days after egg deposition [131], clearly showing the importance of the pupal and pre-emerged adult stage in surviving adverse conditions.

Over the years it has been of general interest to IPM practitioners to develop models that might predict the beginning of flea seasons. A meteorological model was developed to provide an index of weekly activity and an index of cumulative activity over 12 weeks. Only outdoor activity of fleas was considered in developing the model [132]. Keeping a dog indoors or cattle increased the prevalence of cat fleas captured on sticky cards on the floors of households in Yunnan Province, China and thus affected their model [133].

C. felis is known to have numerous endosymbionts, but their role remains largely unknown. In Australia, C. felis had less bacterial diversity than did a native Echidnophaga flea species [67]. Both species were dominated by the endosymbiont Wolbachia. Wolbachia vary among different species of fleas and the practical implications are unknown [134]. An intestinal gregarine, Steinima ctenocephali, neither affected the emergence rates or survival of fleas. Flea larvae with the gregarine developed faster than those without them [135]. A tryponsomatid Leptomonas ctenocephali was found in the digestive tract of adult cat fleas, but its capacity to be pathogenic to fleas or the hosts has yet to be determined [136].

Genomic signatures of convergent adaptation to Alpine environments in three Brassicaceae species

It has long been discussed to what extent related species develop similar genetic mechanisms to adapt to similar environments. Most studies documenting such convergence have either used different lineages within species or surveyed only a limited portion of the genome. Here, we investigated whether similar or different sets of orthologous genes were involved in genetic adaptation of natural populations of three related plant species to similar environmental gradients in the Alps. We used whole-genome pooled population sequencing to study genome-wide SNP variation in 18 natural populations of three Brassicaceae (Arabis alpina, Arabidopsis halleri, and Cardamine resedifolia) from the Swiss Alps. We first de novo assembled draft reference genomes for all three species. We then ran population and landscape genomic analyses with

3 million SNPs per species to look for shared genomic signatures of selection and adaptation in response to similar environmental gradients acting on these species. Genes with a signature of convergent adaptation were found at significantly higher numbers than expected by chance. The most closely related species pair showed the highest relative over-representation of shared adaptation signatures. Moreover, the identified genes of convergent adaptation were enriched for nonsynonymous mutations, suggesting functional relevance of these genes, even though many of the identified candidate genes have hitherto unknown or poorly described functions based on comparison with Arabidopsis thaliana. We conclude that adaptation to heterogeneous Alpine environments in related species is partly driven by convergent evolution, but that most of the genomic signatures of adaptation remain species-specific.

Keywords: Alpine environment Brassicaceae adaptation environmental association genome assembly genome scans.

© 2020 John Wiley & Sons Ltd.


Study system. (a) The three study species from the Brassicaceae family. From left…

Shared signatures of adaptation in…

Shared signatures of adaptation in bayescan outlier genes. Shown is the number of…

Shared signatures of adaptation in…

Shared signatures of adaptation in genes associated with environmental factors. For each environmental…

3 Methods

3.1 Mice

All mice used in the experiments were bred at Australian BioResources (Moss Vale, NSW, Australia) and held at the Garvan Institute of Medical Research in specific pathogen-free environments. The Garvan Animal Ethics Committee approved all mice protocols and procedures.

C57BL/6 (WT) mice were purchased from the Australian BioResources. To generate Lrba −/− mice, an single-guide RNA with the sequence 5′-TTAACTGAGTTGCGGTCACA TGG -3′ (PAM underlined) was microinjected together with Cas9 mRNA into C57BL/6 zygotes. Four of the resulting founder mice were homozygous for an 8 bp deletion eliminating Chr3:86445 392–86 445 399 (Build GRCm38) in exon 37 of the LRBA allele.

HyHEL10-transgenic (SWHEL) mice have been described previously. 45 These mice carry a single-copy VH10 anti-HEL heavy-chain variable region coding exon targeted to the endogenous IgH allele plus multiple copies of VH10-κ anti-HEL light-chain transgene. SWHEL mice were maintained on a CD45.1 congenic (Ptprc a/a ) C57BL6 background. For experiments where SWHEL cells were transferred into LRBA-deficient mice, SWHEL mice were crossed with Rag1-knockout mice 46 to prevent endogenous Igh or Igk gene rearrangement, so that all the developing B cells expressed HyHEL10. This ensured that no lrba +/+ T cells were transferred into LRBA-deficient mice for these experiments.

In addition, SWHEL mice were crossed with lrba −/− mice to generate HyHEL10-transgenic B cells lacking Lrba.

For all experimental interventions animals of both sexes were used in each experimental group and matched for numbers of males and females in test and control groups. Animals were excluded if before recruitment they showed any clinical abnormalities on routine physical examination.

3.2 Bone marrow chimeras and SRBC immunisation

Recipient C57BL/6 Rag1 −/− mice 8–12 weeks old were irradiated with 425 cGy using an X-RAD 320 Biological Irradiator (Precision X-Ray, North Branford, CT, USA). Donor bone marrow was aspirated from femurs, humeri and tibia into B-cell medium comprising RPMI (Gibco, Carlsbad, CA, USA) with 10% heat-inactivated foetal calf serum (Gibco), 2 m m l -glutamine and 100 U/ml penicillin RPMI media (Gibco). At 15 h after irradiation, recipient mice were transplanted with an intravenous injection of 5–10 × 10 6 bone marrow cells, comprising a mixture of 50% from CD45.1 congenic C57BL/6 mice and 50% from C57BL6 (CD45.2) mice that were either Lrba −/− or Lrba +/+ .

Unmanipulated mice 8 weeks old, and bone marrow chimeras 8 weeks after marrow transplantation, were immunised with 2 × 10 8 SRBCs given intravenously.

3.3 Recombinant HEL proteins

Recombinant HEL 3X were made as secreted proteins in Pichia pastoris yeast (Invitrogen, Carlsbad, CA, USA) and purified from culture supernatants by ion exchange chromatography as described previously. 26 , 27 , 45 Proteins were stored in phosphate-buffered saline at 1–2.5 mg ml −1 at −80 °C. Before use samples were thawed and stored at 4 °C for a maximum of 8 months. Upon thawing, protein concentrations were determined by spectrophotometry at 280 nm.

3.4 SRBC conjugation and transfer

HEL proteins were desalted into Conjugation buffer (distilled water with 0.35 m d -mannitol (Sigma, St Louis, MO, USA) and 0.01 m sodium chloride (Sigma)). For this process, PD-10 columns (Amersham, Piscataway, NJ, USA) were equilibrated with 30 ml Conjugation buffer. One hundred micrograms of protein was loaded onto each column and pushed through the column using 2.5 ml Conjugation buffer. For elution of the protein 3.5 ml Conjugation buffer was added and the HEL protein was collected as fractions in the following volumes: 250, 1000, 250, 250 and 250 μl. Protein concentrations of each fraction were determined by spectrophotometry.

For conjugation, SRBCs were washed in 30 ml of phosphate-buffered saline per 6–8 × 10 9 cells and then once in the Conjugation buffer. SRBCs were then resuspended in a final volume of 1000 μl conjugation buffer in a 50 ml Falcon tube containing 10 μg ml −1 of HEL 3X . The solution was mixed on a platform rocker on ice for 10 min. One hundred microliters of 100 mg ml −1 N-(3-dimethylaminopropyl)-N-ethylcarbodimide hydrochloride (Sigma) was then added and the solution was mixed for a further 30 min on ice. Confirmation of successful conjugation was performed by flow cytometric analysis of SRBC using AlexaFluor 647-conjugated HyHEL9 antibody (generated in-house).

Intravenous transfers of 3 × 10 4 SWHEL B cells per recipient mouse together with 2 × 10 8 HEL 3X SRBC as described previously. 26

3.5 Haematology and flow cytometry

Red blood cells and platelet numbers were determined in blood collected from the tail-vein into EDTA (Sarstedt, North Rhine-Westphalia, Nümbrecht, Germany) tubes and analysed on a Sysmex XT-2000iV automated haematology analyser, Kobe, Hyōgo Prefecture, Japan.

On the day of harvest organs were collected into B-cell medium, cell suspensions passed through a 70 μm cell strainer (Falcon, Corning, NY, USA). Fc receptors were blocked with unlabelled anti-CD16/32 (eBioscience, San Diego, CA, USA) before staining. To detect HEL 3X -binding cells, cells were stained with 200 ng ml −1 HEL 3X , followed by AlexaFluor 647-conjugated HyHEL9.

Anti-IgG1-FITC (BD Pharminigen, San Diego, CA, USA) stains were followed by 5% mouse serum before staining for other surface molecules. CD4-BV786 (BD Pharminigen), CD8-APCCy7 (BD Pharminigen), CD62L-PerCPCy5.5 (BD Pharminigen) CD44-FITC (BD Pharminigen) and CD25-PE (BD Pharminigen) were used as surface stains. For the intracellular stains, CTLA-4-APC (eBioscience) and FOXP3-EF450 (eBioscience) cells were first permeabilised using FOXP3 Staining Permeabilisation Kit (eBioscience) according to the manufacturer's instructions. Cells were filtered using 35 μm filter round-bottom FACS tubes (BD Pharminigen) immediately before data acquisition on an LSR II analyser (BD Pharminigen).

Cytometer files were analysed with the FlowJo Software (FlowJo LLC, Ashland, OR, USA).

3.6 Single-cell FACS sorting

Cell suspensions were prepared and GC B cells were identified using flow cytometry. Single-cell sorting into 96-well plates (Thermo Fisher Scientific, Boston, MA, USA) was performed on the FACSAria or FACSAriaIII (BD Pharminigen). B cells from each mouse were analysed individually to ensure that over-representation of one particular clone did not affect the mutation analysis. The VDJH exon of the Hy10 heavy-chain gene was amplified from genomic DNA by PCR, sequenced and analysed as described previously. 27

3.7 Enzyme-linked immunosorbent assay

High-binding plates (Corning, NY, USA) were coated with the indicated Ig isotypes at 5 μg ml −1 (IgG2b (BD Pharminigen), clone: R9-91 IgA (BD Pharminigen), clone: C10-3 IgG1 (BD Pharminigen), clone: A85-1 IgM (BD Pharminigen), clone: 11/41 IgG3 (BD Pharminigen), clone: R2-38 IgG2a(b) (BD Pharminigen), clone: R11-89 IgE (BD Pharminigen), clone: R35-72). Bound serum antibody was quantified using Igκ (BD Pharminigen). Antibody levels were quantified against isotype-specific standards (IgG2b BD, IgA BD, IgG1 BD, IgM BD, IgG3 BD, IgG2a(b) (Southern Biotech, Birmingham, AL, USA) and IgE (BioLegend, San Diego, CA, USA)).

3.8 Viral infection

Mice were infected intravenously with 2 × 10 6 plaque-forming units of LCMV clone 13. T-cell stimulation with LCMV peptide, tetramer staining, surface staining and intracellular cytokine staining were performed as described previously. 47 , 48 , 49 MHC I LCMV tetramers were purchased from the Biomolecular Resource Facility, JCSMR, ANU, ACT, Australia, while the MHC II LCMV GP66–77 tetramer was obtained from the NIH Tetramer Core Facility (Emory University, Atlanta, GA, USA).

3.9 Statistical analysis

GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) was used for data analysis. When the data were normally distributed, two-tailed Student's t-test was performed for analysis. Welch's correction was used if variances were not equal. For all tests P<0.05 was considered as being statistically significant. In all graphs presented error bars indicate mean and standard distribution. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.


We thank the Garvan Institute ABR, GMG and Flow Cytometry facilities for expert animal husbandry, genotyping and cell sorting. This work was supported by NHMRC Project Grant 1108800, NHMRC Program Grants 1016953 and 1113904, NIH Grant U19 AI100627 and NHMRC Fellowship 1081858, and by the Ritchie Family Foundation.

The authors declare no conflict of interest.

LRBA deficiency decreases CTLA-4 on CD4 effector/memory and Tregs. Flow cytometric analysis of spleen cells from age- and sex-matched Lrba −/− (blue) or WT (red) mice at 6 (n=6) or 26 weeks (n=4) of age. (ac) CTLA-4 expression on CD4 memory/effector T cells. (a) Representative flow cytometry plots of permeabilised CD4 + FOXP3 − cells from 6-week-old mice showing % CTLA-4 + CD44 hi cells and histograms of CTLA-4 in the CD4 + CD44 hi CTLA-4 + population in 6- and 26-week- old mice. Lrba +/− are indicated in pale blue. Graphs show (b) the number of CTLA-4 + CD4 + CD44 hi FOXP3 − cells and (c) CTLA-4 mean fluorescence intensity (MFI) in individual mice and arithmetic mean for each genotype and age group. (dg) CTLA-4 expression on CD4 + Treg cells. (d) Representative plots of permeabilised CD4 + cells showing the % FOXP3 + Treg cells in 6-week-old mice. Representative Treg histograms and MFI for: (e) CTLA-4 (f) FOXP3 (g) CD25. Statistical analysis was carried out using t-test: *P<0.05 **P<0.01 ***P<0.001 ****P<0.0001. Data are representative of one experiment.

Absence of immune dysregulation disease in LRBA-deficient mice. (af) Analysis of sex-matched WT (red) and Lrba −/− (blue) mice at 6 weeks of age, showing individual results and means for each group. (ac) Flow cytometric analysis of spleen, showing the numbers of: (a) CD4 + or CD8 + T-cell subsets of central memory (CD62L + CD44 + ), effector memory (CD62L − CD44 + ), naïve (CD62L + CD44 − ) or Tregs (CD4 + FOXP3 + ). (b) B cells (B220 + ) and subsets of transitional 1 (T1, B220 + CD93 + CD23 − ), transitional 2 and 3 (B220 + CD93 + CD23 + ), marginal zone (B220 + CD21 + CD23 − ), mature follicular (B220 + CD93 − CD23 + ) and B-1 (B220 − CD19 + ) B cells per spleen. (c) neutrophils (B220 − MHC II − Ly6G + CD11b + ) and NK lymphocytes (B220 − CD8 − MHC II − NK1.1 + ). (d) CD86 mean fluorescence intensity (MFI) on marginal zone and follicular B cells. (e) Flow cytometric analysis of bone marrow, showing % of lymphocytes that are pro-B cells (B220 + CD43 int ), pre-B cells (B220 + CD43 − IgM − ), immature B cells (B220 int CD43 − IgM + IgD − ), transitional B cells (B220 hi CD43 − IgM + CD24 + IgD lo ) and mature B cells (B220 hi CD43 − IgM + CD24 − IgD + ). (f) Flow cytometric analysis of thymus, showing number of CD8 − CD4 − double-negative (DN), double-positive (DP), CD8 + or CD4 + single-positive, or CD4 + FoxP3 + Treg cells. N=6 per group. Data are representative of one experiment. (g) Titres of IgG2b, IgA, IgG1, IgM, IgG3 Ig2Ga(b) and IgE antibodies in the serum of unimmunised mice, determined by enzyme-linked immunosorbent assay (ELISA). N=10 per group. Representative of two comparable experiments. Statistical analysis was carried out using t-test: **P<0.01.

Decreased peritoneal B-1 B cells in LRBA-deficient mice. Flow cytometric analysis of peritoneal cavity lymphocytes from sex-matched WT (red) and Lrba −/− (blue) mice at 6 (n=6) or 26 (n=4) weeks of age, showing individual results and means for each group. (a and b) Percentage of lymphocytes that are T cells (B220 − IgM − CD23 − CD5 + ), B-2 cells (CD19 − B220 hi ), B-1 cells (CD19 + B220 lo IgM + CD23 − ), B-1a cells (CD19 + B220 lo IgM + CD23 − CD5 + ) and B-1b cells (CD19 + B220 lo IgM + CD23 − CD5 − ). (c and d) Representative flow cytometric plots showing gating IgM, CD23, B220 and CD19 staining for B-1 and B-2 cells and representative CD5 histograms of B-1 cells showing gates on CD5+ B-1a and CD5- B-1b cells from mice at 6 (c) and 26 (d) weeks of age. Statistical analysis was carried out using t-test: *P<0.05 **P<0.01. Data are representative of one experiment.

Cell-autonomous loss of CTLA-4 on LRBA-deficient T cells in bone marrow chimeras. Irradiated Rag1 −/− recipient mice were transplanted with a bone marrow mixture comprising 50% CD45.1 + Lrba +/+ cells and 50% CD45.2 + cells that were either Lrba −/− or Lrba +/+ (WT). One hundred percent CD45.2 + Lrba −/− or Lrba +/+ (WT) marrow was transplanted into a parallel group of Rag1 −/− recipients. At 8 weeks after transplantation, the chimeric mice were immunised with SRBCs three times 3 days apart and blood was analysed by flow cytometry 62 days after the first immunisation. Lines connect paired values for CD45.1 and CD45.2 cells in individual chimeric mice. (a) Percentage of CD45.2 + Lrba −/− (blue) or Lrba +/+ (WT, red) cells among the indicated lymphocyte subsets in individual mixed chimeric mice and arithmetic means: B cells (B220 + ), T cells (CD3 + ), CD4 + T cells, effector CD4 + T cells (CD25 + , CD44 hi ), CD8 + T cells, effector CD8 + T cells (CD25 + , CD44 hi ) and Tregs (CD4 + FOXP3 + ). (be) Analysis of CD4 + FOXP3 − cells. (b) Representative plots of intracellular CTLA-4 and CD44 gated on CD45.1 + (WT) or CD45.2 + (WT or Lrba −/− ) CD4 + FOXP3 − cells. (ce) Analysis of CTLA4 + CD44 + CD4 + FOXP3 − cells, showing: (c) percentage among the CD45.1 + or CD45.2 + subsets of total T cells (d) representative CTLA-4 histograms (e) CTLA-4 MFI values. Dotted lines represent CD45.2+ (Lrba +/+ or Lrba −/− ) cells in mixed chimeras solid lines represent equivalent cells in 100% Lrba +/+ or Lrba −/− chimeras. (fj) Analysis of CD4 + FOXP3 + cells. (f) Representative flow cytometric plots of CD45.2 + CD4 + cells, showing % Tregs. (g) CTLA-4 MFI values for each chimeric mouse. (h) Representative intracellular CTLA-4 histograms. (i) Representative intracellular FOXP3 histograms. (j) Representative CD25 histograms. Statistical analysis was carried out using t-test between mice or paired t-test when cells were from the same chimeric mouse: *P<0.05 **P<0.01 ***P<0.001 ****P<0.0001. N=5 per group. Data are representative of one experiment.

Response of LRBA-deficient mice to chronic systemic viral infection. C57BL/6 Lrba +/+ (WT) (red) or Lrba −/− mice (blue) were infected intravenously with 2 × 10 6 plaque-forming unit (PFU) LCMV clone 13 to induce chronic viral infection. At 20 days after infection, spleen cells were analysed by flow cytometry. (a) Analysis plots of spleens, showing % of lymphocytes that are CD44 hi effector/memory cells in individual mice with arithmetic means. (b and c) Representative flow cytometric plots (b) and anlaysis plots (c) showing % of CD8 + T cells binding MHC I tetramers loaded with LCMV peptides GP33–41 or NP396–404, or the % of CD4 + cells binding MHC II tetramers fused with LCMV peptide GP66–77 in individual mice with arithmetic means. (d) Representative histograms showing TIM3 staining on NP396–404 MHC I tetramer-binding CD8 + T cells in WT and Lrba −/− mice, and MFIs in individual animals for TIM3, CD160 and PD-1. Similar trends were seen with the GP33–41 tetramer. (e) Representative LY6C staining on GP66–77 MHC II tetramer-binding CD4 + T cells, and MFI's in individual animals for LY6C, PSGL1 and PD-1. (f) IFNγ + MFI of the IFNγ + T cells (top) and % TNFα + within the IFNγ + cells. Representative IFNγ and TNFα histograms are shown for the NP396 peptide-stimulated cells. Statistical analysis was carried out using t-test: *P<0.05 **P<0.01. N=5 per group. Data are representative of one experiment. IFNγ, interferon-γ.

Normal GC formation and affinity maturation by LRBA-deficient B cells. CD45.1 congenic C57BL/6 recipient mice, with WT LRBA, were injected intravenously with 30 000 HyHEL10 + SWHEL spleen B cells from Lrba −/− or Lrba +/+ C57BL/6 donor mice. The recipient mice were immunised two times on days 0 and 4 after B-cell transfer with HEL 3X -SRBC, or unconjugated SRBC for a control group of recipients, and spleen cells were analysed by flow cytometry, sorting and single-cell Igh sequencing on day 15. (a) Total Fas hi CD38 − B220 + GC B cells per spleen of individual mice, and arithmetic mean for each group. (b) Donor-derived HyHEL10 + CD45.2 + CD45.1 − GC B cells per spleen. (c) Affinity-matured cells, measured as % donor-derived GC B cells stained brightly with 200 ng/ml HEL 3X . (d) IgG1-switched cells, measured as % of donor-derived GC B cells. (e) Light-zone CD86 + CXCR4 − GC B cells, measured as % of donor-derived GC B cells. (f) CD86 MFI on donor-derived GC B cells. (g) Number of VDJH amino-acid changing or silent nucleotide substitutions per donor-derived GC B cell. (h) Percentage of donor-derived GC B cells with affinity-increasing VDJH mutations S31R, Y53D or Y58F. (i) Percentage of donor-derived GC B cells with substitutions at each VDJH amino-acid position. (j) Co-occurrence of S31R, Y53D and Y58F mutations (rows) in individual cells (columns) sorted from separate recipient mice (boxes). Data are pooled from two independent experiments with comparable results. N=9 mice per HEL 3X -immunised group and four unconjugated controls. Statistical analysis was carried out using t-test: *P<0.05.

Normal GC formation and affinity maturation by LRBA WT B cells in LRBA-deficient recipients. The 30 000 HyHEL10 + SWHEL B cells from Rag1 −/− CD45.1 congenic mice, with WT LRBA, were injected into the circulation of Lrba −/− (n=13, blue symbols) or Lrba +/+ (n=13, red symbols) C57BL/6 recipient mice, so that all T- and B-cell specificities other than the HyHEL10 + B cells were derived from the recipient mice. The recipient mice were immunised two times with HEL 3X -SRBC on days 0 and 4 after B-cell transfer, or unconjugated SRBC for a control group of recipients, and spleen cells analysed by flow cytometry, sorting and single-cell Igh sequencing on day 15. (a) Total Fas hi CD38 − B220 + GC B cells per spleen of individual mice, and arithmetic mean for each group. (b) Donor-derived HyHEL10 + CD45.2 − CD45.1 + GC B cells per spleen. (c) IgG1-switched cells, measured as % of donor-derived GC B cells. (d and e) Relative cell surface of CD86 MFI on (d) all B220 + B cells or (e) donor-derived GC B cells, normalised to mean of Lrba +/+ recipient group in each experiment. (f) Representative plots of donor-derived GC B cells, and gates on light-zone (LZ, CD86 hi CXCR4 lo ) and dark-zone (CD86 lo CXCR4 hi ) GC cells. (g) Relative cell surface CD86 MFI on LZ donor-derived GC B cells, normalised to mean of Lrba +/+ recipient group in each experiment. (h) Number of VDJH amino-acid changing or silent nucleotide substitutions per donor-derived GC B cell. (i) Percentage of donor-derived GC B cells with substitutions at each VDJH amino-acid position. (j) Percentage of donor-derived GC B cells with affinity-increasing VDJH mutations S31R, Y53D or Y58F. (k) Co-occurrence of S31R, Y53D and Y58F mutations (rows) in individual cells (columns) sorted from separate recipient mice (boxes). Data are pooled from two independent experiments with comparable results. Statistical analysis was carried out using t-test: **P<0.01 ***P<0.001.

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