The Endangered Species Challenge: Making Use of Opportunistic Sampling Erin Ehmke, PhD – Research Manager Sarah Zehr, PhD – Data Manager The DLC Colony • The world’s largest living colony of endangered primates

• Currently, 22 species, 11 genera and nearly 250 individual strepsirrhine primates

• No other primate center or zoo maintains a similar diversity of prosimians; smaller colonies elsewhere are largely or entirely unavailable for research.

• In the wild, the 80+ species of lemurs are endemic only to Madagascar

• All lemur species are protected by CITES Appendix I regulations and the Endangered Species Act

• It would be impossible to duplicate the diversity of the DLC colony today.

• 48 year history  long-term data on known individuals with known pedigrees

The Duke Lemur Center conservation research

education hey. a squirrel. Research at the DLC

# in research Species (2013) current colony C. medius (fat-tailed dwarf lemur) 15 19 D. madagascariensis (aye aye) 16 16 E. collaris (collared lemur) 6* 4 E. coronatus (crowned lemur) 11* 10 E. macaco flavifrons (blue-eyed black lemur) 5 14 E. macaco macaco (black lemur) 1 3 E. mongoz (mongoose lemur) 10 11 E. rubriventer (red-bellied lemur) 6 6 E. rufus (red-fronted lemur) 2 2 Eulemur sp. 3 4 H. griseus (grey gentle lemur) 3 3 L. catta (ring-tailed lemur) 40* 37 M. murinus (mouse lemur) 42 48 N. pygmaeus (pygmy slow loris) 1 8 P. coquerli (Coquerel's sifaka) 26 31 Varecia rubra (red ruffed lemur) 19* 15 Varecia variegata (black & white ruffed lemur) 12* 11 TOTAL NUMBER OF ANIMALS1 218 242 PERCENTAGE OF COLONY USED 90.01% Biological Sample Collection

• 48 year history • ~ 10,000 samples • >1000 individuals • >35 taxa

Current Stock

Sample type number species individuals

Blood 1815 28 435

Serum 3215 30 598

Cadavers 400 36 400

Ultra cold tissues 2644 34 552

Urine 582 21 178

RNA tissues 984 22 69 (Non) Model Organisms • Different perspective on biobanking – opportunistic – research process in reverse

• Push imaginations and technology to limit to collect biologically meaningful data consistent with non-invasive policies

• Colony is healthily aged (no introduced disease process)

Opportunistic Sampling

• Routine veterinary exams (blood, serum, urine, feces…)

• Necropsy (major organs, intestinal contents, blood, cadaver…) • Unusual samples (sperm plugs, extracted teeth, amputated fingers…) Anticipate sample types

• What tissue types have been requested before? • When to sample extensively and when to leave intact? • What are current trends in research questions? Diversify sampling

• Ultra-cold banked tissues • RNA-later banked tissues • Frozen cadavers • Formalin fixed samples Document! • Animal identification • Time to freezer • Circumstances of collection • Storage location • Time and circumstance of death • Histopath results; comorbidity • Time of collection • Samples out

Know your samples • What subsets of frozen tissues are acceptable for RNA analysis? • How many times has a particular sample been thawed? • What parts of cadavers are missing? Distribute sparingly

• Store in logical aliquot sizes to minimize freeze/thaw cycles or post-collection handling

• Familiarize yourself with sample amount requirements for common analysis types

• Parcel out cadavers carefully – One cadaver can support a dozen projects; take samples from already damaged ones if possible

• When selecting specimens, send out samples from well-represented individuals if individual ID doesn’t matter Biological Sample distribution (2007 - May, 2014)

2007 2008 2009 2010 2011 2012 2013 2014 All

Cadavers 38 42 26 153 51 62 28 13 413

Blood products 154 374 130 255 230 151 157 42 1493

Fluids (feces, urine, odors) 193 492 153 432 126 138 191 63 1788

Organs 102 89 91 95 113 33 26 76 625

Other (hair, swabs, etc) 4 32 9 21 20 28 108 21 243

Numbers for 2014 are through mid-April Data! Woo hoo! Duke Lemur Center 1966-current 4157 individuals; >30 Taxa Lemurs, Lorises, Galagos, Tarsiers

DLC Database Project: 2012- Direct: DATA VARIABLES Species Individual ID Sex Calculated: Date of Birth Relatedness and Pedigrees Date of Death Breeding seasonality Dam and Sire ID Litter size Housing Birth order Diet Birth and adult body weight Weights Seasonal weight variation Reproductive status Group size Group composition Additional: Sex ratio Gestation length Age at weight Medical records Age at reproduction X-rays Age at death Banked biological samples Date of conception Research participation Temperature at conception Temperature Day length at conception Photoperiod Birth season order Activity (noctural/diurnal) Social systems Phylogeny Variable CMED DMAD EALB ECOL ECOR EFUL EMF EMM EMON ERUB ERUF ESAN

S_N_All_Historic 252 44 40 71 76 65 73 119 126 30 170 22

S_N_M_Historic 122 22 20 38 44 37 38 58 58 18 77 11 S_N_F_Historic Prosimian99 primate22 18 life31 history29 25profiles 34 53generated 65 12 86 11 S_N_All_WildBorn 4 7 6 6 4 6 7 5 18 6 7 5 S_N_M_WildBorn from 2the new3 3Duke 4 Lemur2 Center3 2 Database2 8 3 4 2 S_N_F_WildBorn 2 4 3 2 2 3 5 3 10 3 3 3

S_N_All_CurrentResident (19coming 16 soon1 4 to a 10URL near . 13 you3 !) 11 6 2 1

R_Ratio_MtoF_DLCBirths 1.264 0.75 1 1.28 1.591 1.563 1.037 1.19 1 1.444 0.867 1.125

S_N_All_DLCBorn_Litter 103 28 17 48 48 39 53 72 76 21 133 16

R_Mean_LitterSize 2.19 1 1.65 1.23 1.25 1.13 1.04 1.38 1.03 1.05 1.08 1.06

R_MostCommon_LitterSize 2 1 1,2 1 1 1 1 1 1 1 1 1

R_Max_LitterSize 5 1 4 2 2 2 2 2 2 2 2 2

R_Expected_Gestation_day 60 165 120 120 120 120 120 120 120 120 120 120

R_Peak_Breeding_Month 5 0 12 12 1 12 11 11 12 12 11 1

R_Peak_Breeding_Season 4.5.6 0 1.12 1.11.12 1.12 11.12 11 11.12 1.12 1.11.12 11.12 1.3.12

R_Min_Dam_AgeAtConcep_yr 0.8 4.22 2.57 1.64 1.71 1.39 1.59 1.48 1.78 1.78 1.55 1.61

R_Min_Sire_AgeAtConcep_yr 1.8 3.66 2.75 2.52 2.48 1.74 2.66 0.73 2.92 3.7 1.5 2.03

R_Max_Dam_AgeAtConcep_yr 14.85 26.03 11.31 23.2 17.47 19.18 16.89 15.69 23.6 13.15 22.37 19.28

R_Max_Sire_AgeAtConcep_yr 16.11 28.47 16.31 21.22 19.92 19.18 21.13 14.73 19.69 14.3 24.29 8.43

S_N_All_AdultsWeighed 112 15 8 38 34 24 41 37 58 17 47 17

M_Mean_All_AdultWeight_g 241.29 2649.65 2233.61 2337.04 1647.15 2485.5 2437.45 2467.59 1556.44 2190.57 2289.56 2111.97

M_Mean_All_NeonateWeight_g 13.41 109.93 . 68.48 48.07 73 82.72 70.1 62.14 76.53 77.06 93.6

L_Max_All_Age_yr 28.79 32.37 33.87 32.61 27.42 36 24.37 30.02 34.86 31.5 32.18 31.5

L_Pct_All_InfMort_lt30days 0.3 0.11 0.21 0.25 0.37 0.27 0.27 0.32 0.24 0.36 0.26 0.06

L_Pct_M_InfMort_lt30days 0.16 0.08 0.08 0.22 0.37 0.16 0.18 0.22 0.18 0.38 0.26 0.11

L_Pct_F_InfMort_lt30days 0.23 0.13 0.23 0.24 0.27 0.31 0.37 0.33 0.26 0.33 0.21 .

O_N_All_Biosample_Individuals 43 34 5 35 34 7 47 24 48 13 32 11 Brain Weight vs Individual body weight

R2=0.845 Organ Weight vs Individual body weight

R2=0.845

R2=0.793

R2=0.409

R2=0.348 Organ Weight vs Individual body weight

R2=0.845

R2=0.793

R2=0.409

R2=0.348

Identify abnormal tissues; categorize as healthy vs diseased; correct errors On-going challenges

• Limited quantity; unpredictable • Decades old samples • Unused samples • Back-up storage • Permits • Fee structure

Can an endangered species be a model organism? The mouse lemur: An ideal organismal system

• Small body size (60-80g)

• Reproduce rapidly (60 day gestation) – reproductive age = 0.75 - 7 years – average litter = 2 offspring

• They are primates, and thus much more closely related to humans than are mice or other small model organisms.

• They also develop age-related symptoms of dementia, similar to Alzheimer's in humans.

• They mature and age rapidly relative to other non-human primate models.

The mouse lemur: An ideal organismal system

• The DLC houses the only breeding and research colony of mouse lemurs in North America.

• The live animal colony, known pedigree lines, data and tissue repositories have been vital to NIH-supported research • neurobiology, aging, epigenetics, retroviral and genomic evolution, disease transmission, physiology, cognition, sensory integration and cancer biology

• Use of live-animal MRI and high-resolution post-mortem CT scans to characterize basic neuroanatomical landmarks and brain areas to interpret the anatomy, development, function and aging of the primate brain.

• Map the connections (the "connectome") in the lemur brain for comparison to that of humans and characterize age-related dementia in primates such as Alzheimer's disease. Collaborators Photo credits Sarah Zehr David Haring Anne Yoder David Brewer DLC animal staff

Lemurs!

Funding