Genomics and morphometrics reveal the adaptive evolution of pikas

Pikas(Lagomorpha: Ochotonidae) are small mouselike lagomorphs. To investigate their adaptation to different ecological environments during their dispersal from the Qinghai-Xizang(Tibet) Plateau(QTP), we collected 226 pikas and measured 20 morphological characteristics and recorded habitat information. We also sequenced the genome of 81specimens, representing 27 putative pika species.The genome-wide tree based on 4?090 coding genes identified five subgenera, i.e., Alienauroa, Conothoa,Lagotona, Ochotona, and Pika, consistent with morphometric data. Morphologically, Alienauroa and Ochotona had similar traits, including smaller size and earlier divergence time compared to other pikas.Consistently, the habitats of Alienauroa and Ochotona differed from those of the remaining subgenera. Phylogenetic signal analysis detected 83 genes significantly related to morphological characteristics, including several visual and hearingrelated genes. Analysis of shared amino acid substitutions and positively selected genes(PSGs) in Alienauroa and Ochotona identified two genes, i.e.,mitochondrial function-related TSFM(p.Q155E) and low-light visual sensitivity-related PROM1(p.H419Y).Functional experiments demonstrated that TSFM-155E significantly enhanced mitochondrial function compared to TSFM-155Q in other pikas, and PROM1-419Y decreased the modeling of dynamic intracellular chloride efflux upon calcium uptake. Alienauroa and Ochotona individuals mostly inhabit different environments(e.g., subtropical forests) than other pikas, suggesting that a shift from the larger ancestral type and changes in sensory acuity and energy enhancement may have been required in their new environments. This study increases our understanding of the evolutionary history of pikas.

Plateau pikas near roads are bold and silent when facing a potential predator

Human disturbance,particularly road traffic,is one of the greatest threats to wildlife.Considering the association between alerting behavior and the survival of animals,it is important to study the effects of road traffic on alerting behavior of wildlife.Previous studies assessing the shortterm impact of road traffic on alerting behavior of wildlife have focused on vigilance distances.However,studies on the use of alarm calls are scarce,and it is unclear whether such behavioral responses change after repeated exposure to road traffic.We assessed the alerting behavior of plateau pikas(Ochotona curzoniae)who were near or far from roads when facing a potential predator.We found that pikas near roads exhibited shorter vigilance and tolerance distances,and produced fewer alarm calls than those relatively far away from roads.Furthermore,both vigilance and tolerance distances of plateau pikas were significantly positively correlated with the distance from the burrow to the road.Road traffic reduced antipredator responses and shaped alerting behavior;that is,pikas near roads were bolder and more silent compared to those far away from roads.Our findings suggest that increasing urbanization will have corresponding effects on animal behavior,which may have significant fitness effects in the future.

Seasonal variations in the composition and functional profiles of gut microbiota reflect dietary changes in plateau pikas

Seasonal variations in gut microbiota of small mammals and how they are influenced by environmental variables are relatively poorly understood.We sampled 162 wild plateau pikas(Ochotona curzoniae)in 4 seasons over 2 and a half years and recorded the air temperature,precipitation,and nutrient content in edible vegetation at the sampling site.After conducting 16S rRNA and shotgun metagenomic sequencing,we found that the highest alpha diversity,the relative abundance of Firmicutes,and the simplest co-occurrence network occurred in winter,whereas the highest relative abundance of Proteobacteria and the most complex network structure were observed in spring.The highest relative abundance of Verrucomicrobiota and Spirochaetota was seen in summer and autumn,respectively.Air temperature,precipitation,and the contents of crude protein,crude fiber,and polysaccharide in vegetation had significant effects on the seasonal changes in gut microbiota.Diet contributed more to microbial variation than climatic factors.Metagenomic analysis revealed that the amino acid metabolism pathway and axillary activity enzymes were most abundant in summer,while abundance of carbohydrate-binding modules and carbohydrate esterases was highest in spring.These microbial variations were related to the changes in dietary nutrition,indicating that gut microbiota of plateau pika contribute to the efficient use of food resources.This study provides new evidence of how external environmental factors affect the intestinal environment of small mammals.

Evolution for extreme living:variation in mitochondrial cytochrome c oxidase genes correlated with elevation in pikas(genus Ochotona)

The genus Ochotona(pikas)is a clade of cold-tolerant lagomorphs that includes many high-elevation species.Pikas offer a unique opportunity to study adaptations and potential limitations of an ecologically important mammal to high-elevation hypoxia.We analyzed the evolution of 3 mitochondrial genes encoding the catalytic core of cytochrome c oxidase(COX)in 10 pika species occupying elevations from sea level to 5000 m.COX is an enzyme highly reliant on oxygen and essential for cell function.One amino acid property,the equilibrium constant(ionization of COOH),was found to be under selection in the overall protein complex.We observed a strong relationship between the net value change in this property and the elevation each species occupies,with higher-elevation species having potentially more efficient proteins.We also found evidence of selection in low-elevation species for potentially less efficient COX,perhaps trading efficiency for heat production in the absence of hypoxia.Our results suggest that different pika species may have evolved elevation-specific COX proteins,specialization that may indicate limitations in their ability to shift their elevational ranges in response to future climate change.

The Influence of Fence on Body Mass and Population Dynamics of Plateau Pika

To determine the influence of the fence on plateau pika (Ochotona curzoniae), we measured the body mass, breeding status, population densities, using live-trapping and line-transect sampling in April and September 2006. In April, body mass of both male and female plateau pika inside of fence were significant higher than that outside of fence (♀: F = 6.583, df = 1.20, p = 0.019;♂: F = 6.49, df = 1.17, p = 0.021). The ratio of spermary drop of an adult male was also higher (92.31% vs 57.14%). In September, body mass of adult male in both study sites showed no significant difference (F = 0.001, df = 1.23, p = 0.975), but were all significant higher than that of April (F = 121.713, df = 1.22, p < 0.001;F = 105.819, df = 1.20, p < 0.001). In April, the population densities of both study sites showed on significant difference (F = 2.388, df = 1.38, p = 0.131), but the population density inside of fence was much higher than that outside of fence (F = 7.534, df = 1.38, p = 0.009) in September. Our results show that the changes of quality and quantity of food that available to plateau pika in winter, can depress the range of reducing body mass of plateau pika, make breeding season ahead, and then promote the increase of population.

Degradation and recovery of alpine meadow catenas in the source zone of the Yellow River,Western China

Process interactions on catenas have supported grazing adapted ecosystems and sustained biodiversity values in the source zone of the Yellow River in western China for millennia.In recent decades,anthropogenic disturbance and climate change have threatened the integrity of these systems,impacting upon environmental values and their capacity to sustain local livelihoods.Collaborations between local experts and a team of international researchers during a workshop and field excursion to this area in July 2019 developed a cross-disciplinary,process-based model of alpine meadow catenas.This paper relates the contemporary health of these grassland-wetland systems to their‘best achievable state’under prevailing boundary conditions,differentiating stages of degradation and recovery in relation to climate and land use changes.Recovery is underway for alpine meadow catenas at Maqin.Reduced land use pressures(stocking rates)and longer growing seasons have enhanced grasslandwetland conditions.However,recovery prospects are limited for local areas of extremely degraded grasslands(heitutan),as breached abiotic thresholds have resulted in soil and nutrient loss and reduced capacity for water retention.While lagomorphs and rodents act as ecosystem engineers when alpine meadows are in a healthy state,irruptions locally increase the proportion of bare ground and inhibit recovery potential.Management options that support recovery of alpine meadows are presented for differing stages of degradation.

Influences of pika and simulated grazing disturbances on bare patches of alpine meadow in the Yellow River Source Zone

Bare patches in alpine meadow are the main manifestation of its degradation.The change of bare patches in an alpine meadow in the Yellow River Source Zone during 2018-2019 was studied in relation to the disturbances caused by plateau pika(Ochotona curzoniae)population and simulated grazing via artificial mowing both independently and interactively.The disturbance was set at three levels of high,medium and no disturbance(control group).Bare patches were mapped by from unmanned aerial vehicle(UAV)images with fine resolution of 1 cm obtained in August 2018 and August 2019 in ArcGIS.The results showed that the total area of bare patches decreased by 112.05 m2 in sub-plots devoid of pika disturbance but increased by 126.37 m2 in other subplots.The highest rate of increase is 89.02%.The individual effect of pika exceeds the joint effect of pika and mowing disturbances.The sole effect of mowing is lower than the joint effect of pika disturbance and intensive mowing,but higher than the joint influence of pika disturbance and moderate mowing.Strong pika disturbance(14 per sub-plot)caused the influence of mowing from moderate to intensive to increase by five-fold.The area of bare patches treated with moderate mowing and no pika disturbance decreased at the highest pace(-37.22%).Intensive mowing and medium density pikas(100 pikas/ha)are considered the thresholds at which the bare patches start to expand.Even if the meadow is mowed at the medium and high intensity,the area of bare patches can be significantly reduced if plateau pika population is controlled to a low level.ANOVA analysis and longterm macro-scale satellite-derived results reveal that pika disturbance is more important in causing the bare patches to change than simulated grazing.Therefore,it is more important to control the number of pikas than to reduce grazing intensity to prevent the expansion of bare patches in the degraded alpine meadow in the study area.

Equally Knocking on the Door of Medical Breakthroughs: Safety and Efficacy of a New Anti-Cancer Drug Pamica (PICNH₂Ca) for Late Stage Cancer Patients

The composition of Pamica (PICNH2Ca2) is dsRNA polyI:C, non-biotics amino compounds and CaCl2. It is the second generation noncytotoxic product of PICKCa (PIKA) adjuvant which was found effective in phase II clinical trial for PICKCa rabies vaccine and phase I trial for PICKCa hepatitis B vaccine in Singapore. With their knowledge and consent, cancer patients, regardless of their tumor types or performance status (ECOG above 5 or ECOG > 20), who were unresponsive to standard treatment, were treated free of charge with Pamica by injection or nasal spray. The primary results indicated that Pamica had no side effect except for a few cases of local pain in the site of injection and appeared to markedly improve cancer patients’ appetite, physical strength, quality of life and extended patients’ life for months and counting. These preliminary findings underlined promise of this potentially life-saving drug. In the hope of cooperating with the proper authorities, companies, and hospitals to benefit patients at the earliest opportunity, we share our findings in this paper.

Escherichia coli Expression of Lactate Dehydrogenase C Gene of Black-Lipped Pika in Western Sichuan Plateau

[ Objective] To express lactate dehydrogenase C （LDH-C） gene in prokaryotic system and then purify the recombinant protein. E Meth- od] The LDI-I-C gene was amplified from black-lipped pika（ Ochotona curzoniae） by RT-PCR and ligated to the expression vector pET-32a. The E. coli BL21 （DE3） carrying the LDI-I-C gene was obtained and induced by IPTG （isopropy-beta-D-thiogalactoside）. The expression products were analyzed by SDS-PAGE and purified by affinity chromatography. L Result] An approximately 1.0 kbp band appeared in the RT-PCR products as the- oretically expected. As evidenced by PCR and double enzyme digestion, an approximately 1.0 kbp fragment appeared, which indicated successful construction of expression vector. As analyzed by SDS-PAGE, a fusion protein with molecular weight slightly larger than 45 kDa was expressed in form of inclusion body. And very pure fusion protein was obtained by nickel ions-charged affinity chromatography. [ Coaclusion] The LDH-C gene of black-lipped pika has been cloned and expressed in E. coil

THE PIKA IS OUR FRIEND

Soon a furry-faced head with bright eyes and rounded ears will pop up and peer out.You are meeting the plateau pika. Its Tibetan name is abra and its scientific name Ochotona curzoniae.

Alpine grassland degradation intensifies the burrowing behavior of small mammals:evidence for a negative feedback loop

Globally,grassland degradation is an acute ecological problem.In alpine grassland on the Tibetan Plateau,increased densities of various small mammals in degraded grassland are assumed to intensify the degradation process and these mammals are subject to lethal control.However,whether the negative impact of small mammals is solely a result of population size or also a result of activity and behavior has not been tested.In this study,we use plateau pika as a model to compare population size,core area of colony,and the number of burrow entrances and latrines between lightly and severely degraded grassland.We test whether the alleged contribution of pika to grassland degradation is a result of increased population size or increased burrowing activities of individuals in response to lower food abundance.We found that grassland degradation resulted in lower plant species richness,plant height,and biomass.Furthermore,the overall population size of pika was not significantly affected by location in lightly and severely degraded grassland.However,pika core areas in severely grassland degradation were significantly larger and had significantly higher densities of burrows and latrines.Our study provides convincing evidence that habitat-induced changes in the behavior of small,burrowing mammals,such as pika,can exacerbate grassland degradation.This finding has significant implications for managing small mammals and restoring degraded grassland ecosystems.

Altitude-dependent metabolite biomarkers reveal the mechanism of plateau pika adaptation to high altitudes

The harsh environment in the Tibetan plateau,the highest place in the world,poses thermoregulatory challenges and hypoxic stress to animals.The impacts of plateau environment on animal physiology and reproduction include external factors such as strong ultraviolet radiation and low temperature,and internal factors such as animal metabolites and gut microbiota.However,it remains unclear how plateau pika adapt to high altitudes through the combination of serum metabolites and gut microbiota.To this end,we captured 24 wild plateau pikas at the altitudes of 3400,3600,or 3800 m a.s.l.in a Tibetan alpine grassland.Using the machine learning algorithms(random forest),we identified five biomarkers of serum metabolites indicative of the altitudes,that is,dihydrotestosterone,homo-l-arginine,alpha-ketoglutaric-acid,serotonin,and threonine,which were related to body weight,reproduction,and energy metabolism of pika.Those metabolic biomarkers were positively correlated with Lachnospiraceae_Agathobacter,Ruminococcaceae,or Prevotellaceae_Prevotella,suggesting the close relationship between metabolites and gut microbiota.By identifying the metabolic biomarkers and gut microbiota analysis,we reveal the mechanisms of adaptation to high altitudes in plateau pika.

The genomic characteristics and pathogenicity of a mammalian orthoreovirus within a new lineage from wild pika in plateau

Emerging and re-emerging viruses from wild animals have seriously threatened the health of humans and domesticated animals in recent years.Herein,we isolated a new mammalian orthoreovirus(MRV),Pika/MRV/GCCDC7/2019(PMRV-GCCDC7),in the Qinghai-Tibet Plateau wild pika(Ochotona curzoniae).Though the PMRVGCCDC7 shows features of a typical reovirus with ten gene segments arranged in 3:3:4 in length,the virus belongs to an independent evolutionary branch compared to other MRVs based on phylogenetic tree analysis.The results of cellular susceptibility,species tropism,and replication kinetics of PMRV-GCCDC7 indicated the virus could infect four human cell lines(A549,Huh7,HCT,and LoVo)and six non-human cell lines,including Vero-E6,LLCMK2,BHK-21,N2a,MDCK,and RfKT cell,derived from diverse mammals,i.e.monkey,mice,canine and bat,which revealed the potential of PMRV-GCCDC7 to infect a variety of hosts.Infection of BALB/c mice with PMRVGCCDC7 via intranasal inoculation led to relative weight loss,lung tissue damage and inflammation with the increase of virus titer,but no serious respiratory symptoms and death occurred.The characterization of the new reovirus from a plateau-based wild animal has expanded our knowledge of the host range of MRV and provided insight into its risk of trans-species transmission and zoonotic diseases.

Coupled effect of climate change and human activities on the restoration/degradation of the Qinghai-Tibet Plateau grassland

Climate change(CC)and human activities(HA)are the main reasons for the restoration/degradation of the Qinghai-Tibet Plateau(QTP)grassland.Many related studies have been conducted thus far,but the impact mechanism of CC coupled with HA on QTP remains unclear.We summarized the two main coupling factors in recent years(specifically,in the past five years)and obtained the following conclusions.(1)CC and HA have positive and negative effects on the QTP grassland ecosystem.CC primarily affects grassland ecology through temperature,humidification,and extreme climate,and HA mainly affects ecosystems through primary,secondary,and tertiary industries and restoration measures.(2)CC coupled with HA affects soil,plants,animals,and fungi/microbes.CC makes the snow line rise by increasing the temperature,which expands the zone for HA.CC also restricts HA through hydrological changes,extreme climate,and outbreak of pikas and pests.Simultaneously,measures are implemented through HA to control and adapt to CC.Hence,the grassland ecosystem is comprehensively influenced by CC and HA.(3)The grassland ecosystem dynamically adapts to the disturbance caused by CC and HA by changing its physiological characteristics,distribution range,diet structure,community structure,and physical state.Simultaneously,it responds to environmental changes through desertification,poisonous weeds,rodent outbreak,release of harmful gases,and other means.This work can be used as a reference for the sustainable development of the QTP grassland.

PIKA as an Adjuvant Enhances Specific Humoral and Cellular Immune Responses Following the Vaccination of Mice with HBsAg plus PIKA

An adjuvant is usually used to enhance the immune response induced by vaccines. The choice of adjuvant or immune enhancer determines the effectiveness of the immune response. Currently, aluminium （Alum, a generic term for salts of aluminium） is the only FDA-approved adjuvant. Alum predominantly induces the differentiation of Th2 cells and thus mediates an antibody immune response. Therefore, there is an urgent need for new adjuvants that enhance not only humoral but also cellular immune responses. In the present study, we demonstrates that PIKA （a stabilized dsRNA） as an adjuvant directly induces the activation and the proliferation of both B and NK cells in vitro. Injection of PIKA into mice results in the production of cytokines in vivo. In addition, the study demonstrates that PIKA promotes the maturation of bone marrow-derived dendritic cells （BMDCs） including up-regulation of the co-stimulatory molecules CD80, CD86 and CD40, and the induction of cytokines such as IL-12p70, IL-12p40 and IL-6. Importantly, after immunization of mice with HBsAg plus PIKA, the presence of PIKA enhances the titers of HBsAg-specific IgG and HBsAg-specific IFN-γ production. These results demonstrate that PIKA as an adjuvant can promote both humoral and cellular immune responses. These might have an implication in applying PIKA as an adjuvant to be used in the design and development of both therapeutic and preventive vaccines, and used in the clinical study.

The plateau pika has multiple benefits for alpine grassland ecosystem in Qinghai–Tibet Plateau

The plateau pika(Ochotona curzoniae)has been engaged in pest control for many years based on the logic that the plateau pika is responsible for alpine grassland degradation and competes with livestock for forage resources.However,the plateau pika is an important species which has multiple benefits in Qinghai-Tibetan Plateau,as it(i)makes burrows that are the primary homes for lizards and a variety of small birds;(ii)creates microhabitat disturbance that promotes the increase of plant species richness;(iii)serves as the principal prey for almost all of the plateau predator species;and(iv)contributes positively to the dynamic equilibriumof alpine ecosystems.

Brief report on the status of Kozlov’s pika,Ochotona koslowi(Büchner),in the east Kunlun mountains of China

Koslov’s pika,Ochotona koslowi(Büchner),was surveyed in western China.This endangered species is poorly known.We determined that it is a burrowing pika,and lives in social family groups at high densities.It primarily occupies high-elevation cold grassland vegetation.It appears to have a high reproductive rate.More emphasis should be placed on research into this unique species in China.