CALIFORNIA STATE UNIVERSITY SAN MARCOS

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PROJECT SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

MASTER OF SCIENCE

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BIOTECHNOLOGY

PROJECT TITLE: A method to estimate relative telomere length in 27 taxonomically diverse captive avian species

AUTHOR: Haleigh Wood

th DATE OF SUCCESSFUL DEFENSE: April 26 20 I 8

THE PROJECT HAS BEEN ACCEPTED BY THE PROJECT COMMITTEE IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOTECHNOLOGY.

Dr. Betsy Read 1::>~ ~ o ~r~.A 0412611 s PROJECT COMMITTEE CHAIR SIGNATUU:· DATE

Dr. Thomas Jensen ~~6/18 PROJECT COMMITTEE MEMBER SIGNATURE~ DATE

Mrs. Patricia Byrne ~,:1,:tv'1. 04/26/18 PROJECT COMMITTEE MEMBER IGNATlJf, DATE Executive Summary

A method to estimate relative telomere length in 27 taxonomically diverse avian species

Haleigh Wood Telomeres are found at the ends of chromosomes and serve as a protective barrier to prevent damage during DNA replication. Telomeres shorten with each cell replication until they reach a critical length, at which point apoptosis occurs leading to mortality. Increased long-term stress has been shown to cause an increased rate of telomere loss (Boonekamp et al 2014). Previous studies have developed methods relating relative telomere length (RTL) and relate it to chronological age and/or life expectancy in avian species through the use of real-time quantitative polymerase chain reaction (qPCR). However, methods that work on multiple species have not yet been developed. A universal method to determine the relative telomere length across a broad range of species would require primers to amplify a control gene from genomic DNA (gDNA) that are highly conserved. The availability of such a primer set eliminates the need to design primer sets for each new species tested. Herein, we tested the well conserved 18S rRNA for this purpose. We isolated and tested red blood cell gDNA from 157 individuals in 27 different species covering12 phylogenetic orders (Galliformes, Anseriformes, Phoenicopteriformes, Eurypygiformes, Columbiformes, Cuculiformes, Gruiformes, Accipitriformes, Bucerotiformes, Coraciiformes, Psittaciformes, Passeriformes). Five sets of 18S primers were designed and tested for efficiency at the telomere annealing temperature (56°C) determined in previous studies.

The relationship of relative telomere length and chronological age was examined in six species, each with 6 to 50 samples by calculating the coefficient of determination (R2). For the 27 species tested, the average qPCR efficiency of the 18S primers was 94.3% ± 5.9, with efficiencies ranging from 85.2% to 107.6%. R2 values were 0.07 in the nene (Branta sandvicensis), 0.003 in the alala (Corvus hawaiiensis), 0.17 in the Chilean flamingo (Phoenicopterus chilensis), 0.41 in the blue and gold macaw (Ara ararauna), 0.26 in the blue-bellied roller (Coracias cyanogaster) and 0.008 in the greater flamingo (Phoenicopterus roseus). Although we successfully developed a technique to determine RTL in a broad range of avian species, we did not find a relationship between chronological age and RTL for any of the six species, suggesting telomere dynamics in captive populations may be different than those found in the wild.

This study provides a tool that can be used to measure the effects of long-term stress on relative telomere length in captive individuals from a broad range of avian species. Care managers can use this information when determining which individual is most suited for breeding or reintroduction. A method to estimate relative telomere length in 27 taxonomically diverse captive avian species

San Diego Zoo Institute for Conservation Research Haleigh Wood

Committee Members: Dr. Betsy Read, California State University San Marcos Dr. Thomas Jensen, San Diego Zoo Institute for Conservation Research Mrs. Patricia Byrne, San Diego Zoo Institute for Conservation Research Telomere length shortens with each cell replication.

https://www.bioscience.pk/topics/genetics/item/827-telomere-indicator-of-physiological-age Multiple factors influence the rate of telomere loss.

Poor diet

Environmental Oxidative stress stress

Chronological Telomere Genetic age Shortening inheritance Each method offers certain strengths and weaknesses.

Method Strengths Weaknesses Telomere Restriction Provides an absolute Not efficient for large Fragment telomere length sample sizes Time-consuming procedure Expensive Flow-FISH Can be used in large Time-consuming procedure sample sizes Provides absolute telomere Expensive length qPCR Can be used in large Provides relative amount sample sizes of telomere present. Cost efficient Time-sensitive The main goal of our research was to develop a tool for the management of our captive bird population.

• Necessities: • Uses: – Inexpensive – Estimation of – Time-efficient chronological age and

– Readily available life expectancy equipment – Evaluation of long term

– High throughput stress effects – – Applicable to a wide Choosing individuals for breeding/reintroduction range of species

The methodology chosen for estimating relative telomere length was based on speed and simplicity.

Test Age vs. Collection of Reference Verify Relative Blood gDNA Telomere Primer Design Isolation Gene Telomere Samples Amplification Efficiency Length 18S primer set 4 showed the greatest efficiency at Tan of 56°C.

ladder 1 2 3 4 5 ladder

500 bp

100 bp Nene BB roller Nene BB roller Nene BB roller Nene BB roller Nene BB roller Kenya crested guineafowl Mountain bamboo partridge Cabot’s tragopan Nene Greater flamingo Chilean flamingo Kagu

Domestic pigeon Insular crowned pigeon Jambu fruit dove Crested cuoa Guam rail Harpy eagle Cape vulture

California condor Sulawesi hornbill Blue-bellied roller Micronesian kingfisher Blue and gold macaw Edward’s fig parrot Black-cheeked love bird

Rainbow lorikeet Pesquet’s parrot Scarlet macaw Alala Eastern blue-winged minla Javan cochoa http://currents.plos.org/treeoflife/ article/reweaving-the-tapestry-a- supertree-of-birds/ 18S reported an average efficiency of 94.3±5.9 for all 27 species. Galliformes Anseriformes Phoenciopteriformes Eurypygiformes

Columbiformes Cuculiformes Gruiformes Accipitriformes

Bucerotiformes Coraciiformes Psittaciformes Passeriformes

Telomere amplification was achieved in all 27 species. We found no significant correlation between chronological age and relative telomere length.

Many factors could possibly lead to stress in captivity.

• Sounds • Lighting • Substrate • Cage space • Diet • Breeding • Hierarchy

• Inability to escape stressors. Relative telomere length may be a better predictor of life expectancy rather than chronological age.

Boonekamp et al. 2014 We developed a standard method of determining relative telomere length in avian species.

This has the potential to become a management tool to be used across zoos: – Simple and inexpensive method – Readily accessible equipment – Time-efficient Future research: – Life expectancy – Effects of long-term stress References

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