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NROC34 Lec 8.docx

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Karen Williams

NROC34 Lec 8 - Poster party – print out poster, ask people about their posters - Review: due APRIL 5 TH – review of High Background … o First few chapters of the Animal behaviour book – scientists, scholars, naives and fools, o Scientific writing by Goat and Swan – how to write about science and how science is down  Butterflies and Turtles o 2 articles: Merlin et al o Be familiar with the process of reading articles  In graduate courses, talk about how article was done and findings o What is the article about? Merlin et al 2009  Organisms  Monarch Butterflies o How do monarch butterflies find their way to their migrating grounds in Mexico? o How do they know when to leave and where to go? o Looking at them up close: see that they find their way to Mexico by some part of their anatomy that is necessary o How do animals know when to leave on migratory journeys? o Monarch butterfly migration:  Quantitative analyses of migration: flight simulator  Physiological bases: Juvenile hormone, circadian timing  Neuroanatomical basis: cry1 and cry2; per, timeless expression; antennae and the sun compass  Monarch butterfly migration o Autumn migrants use the same overwintering ground every year  Leave in autumn o Sunlight seems to be important – perhaps antennae are involved o Overwintering area in Mexico is 3600 km away o Because they are going all the way to Mexico, probably something in the brain is necessary or eyes – first initial idea o Overwinter in a reproductive diapause  Physiologically, are NOT ready to reproduce o Short days in September are perceived by monarch butterflies which triggers diapause  If perceiving amount of sunlight in a day, that sets of a whole different physiological trajectory  Male declining ejaculatory duct mass and increases in female photoperiod- induced diapause both describe monarch butterflies’ changing reproductive status with season  Cease reproduction  Coincide with their leaving for Mexico o As they leave for Mexico, notice less sunlight and stop reproducing  How to investigate the genetic basis of butterfly migration? o Late summer generation makes autumn migration to Gulf coast o To describe behaviour: at the timing of behaviour (lake, summer, autumn)  Individuals that move south are 3-5 generations away from the generation that previously occupied that site o See monarch butterflies in areas that are warmer – differs on the time of year  Finding a candidate gene o Identify and categorize behaviour of interest   nominate a candidate gene from literature or database searches  background research on gene of interest   candidate gene not available or feasible  measure changes in gene expression using cDNA microarray  clon candidate gene in your organism   clone candidate gene in your organism  --> candidate gene not available or feasible  measure changes in gene expression using cDNA microarray  Gene expression o Took out brains of monarch butterflies (300) and looked for Expressed Sequence Tag (EST) to do gene expression profile o EST database  candidate gene approach  gene expression profile  Monarch Butterfly Migration o Know 2 things about behaviour:  Travel really far (overwinter in Mexico 3600 km away)  Overwinter in a reproductive diapause  Triggered by short days in September  Butterflies in diapause are characterized in males by low ejaculatory duct weight and in females by low numbers of mature oocytes and low ovary weight o Both effects of LOW JH  Candidate genes important for Monarch migration o Some sort of hormone related to low reproduction (juvenile hormone)  Known for breaking diapause o Should look at movement and orientation that Monarchs may be using  Locomotion  Butterfly collections o Can collect migrant butterflies only in Aug, Sept, Oct o Collect both male and female and take brains out  Differences in transcript levels of some genes o JH associated genes: allatotropin, JHAMT, and takeout more highly expressed in SUMMER butterflies o Locomotion gene turtle is more highly expressed in MIGRANT butterflies o Other locomotion associated genes – foraging and single minded are expressed at the SAME LEVELS in both SUMMER and MIGRANT butterflies o Reproductive diapause, JH associated genes and takeout  All JH associated genes  More highly expressed in SUMMER butterflies  Expect those in summer to have higher amounts which is TRUE o Summer butterfly brains –  Black bars are higher for allatotropin, JHAMT, and takeout  Significantly higher mRNA levels in SUMMER butterflies o Turtle was the only ones more highly expressed in MIGRANT butterflies (more mRNA in migrant butterflies)  Gene associated with locomotion is more highly expressed  Perhaps the light going into something in eyes is changing the reproductive physiology in diapause o In brain of the flies, get an integration of thermoperiodic cues and other environmental cues  Such as food quality (eat milkweed) – this is the limit of the butterflies  Might change migratory behaviour o All of these cues could be integrated in the brain of the butterfly  Genes and rhythms o Photoperiodic activity rhythm that is changed with the amount of light in the day o Changes circannaul rhythm – activity that happens within a year  Could be based on o Circadian rhythms – 24 hr cycle of behaviour that expresses itself independent of environmental changes  Clock genes as candidate genes  Basis of next part of candidate genes  Proximate mechanisms o Where the sun is in the sky  Sun    UV polarized light  dorsal tim  central complex  motor system  motor output   PL  central complex  motor system  motor output o Other proximate mechanisms that have to do with diapause o Amount of light perceived at opsins  Sun   Opsins (PL)  temperature other factors? (PI)  decrease in insulin-like peptides  corpora cardiac corpora alata  decrease in JH  o  gonad – decrease reproduction o  soma – increase longevity o Have a movement and a productive physiology mechanism  Candidate genes important for Monarch migration o Candidate genes within the EST database were those associated with:  Diapause – genes regulated by JH known to be important in reproduction and breaking diapause  Locomotion – genes important more movement and orientation since Monarchs use a sun-compass for navigation  Circadian rhythms – genes important in the Drosophila clock mechanism – period, timeless, clock, cycle o Had diapause candidates, locomotion and circadian candidates  If involved in migrant behaviour, should see differences  Differences in amount of circadian gene level at the time of day  Clock genes cycle? o PER and TIME (timeless)  Per is degraded during light o Prediction: if circadian clock is important in Monarch migration, then clock genes should be expressed rhythmically in the Monarch brains o Zeitgeber o In a day of 24 hrs, have a period of light and darkness – expect to see different levels of mRNA associated with presence of light and presence of darkness o Results:  For PER gene – increase in the dark  For TIM – in light is LOW, in dark it is HIGH  In cry1 - Low in the light, peaks at end of darkness  Cry2 – not much cycling  Clock genes cycle? o In monarch butterfly brain, drosophila genes also cycle in monarch brains o Clock proteins PER, TIM and CRYPTOCHROME (CRY) are found in the central brain o Cryptochrome – a blue-light photoreceptor  Humans also have and plants  Very low light photoreceptor  Found in the brain of monarch butterflies  Summary o Candidate genes change between summer and winter o Genes expressed in the brains of Monarch butterflies: transcriptome  Found candidate genes that were changed between summer and migrant butterflies  Genes and rhythms  Clock genes identified in Monarch EST database period Clock gene timeless Clock gene Clock Transcription factor Cycle Transcription factor Cryptochrome photoreceptor Cryptochrome 2  Where is the clock? o Came into the study thinking that the brain had the location o Everything suggested that the clock was in the brain of monarch butterflies o Usually antennae are used for olfaction – perhaps flies navigate in olfactory plume or pheromone  Usually get navigating abilities (males looking for females) o If antennae are important, need to know how to describe behaviour and ask if you remove antennae would you see a difference in behaviour? o Tether monarch butterfly and see where it moves  Expect SW direction for migration o Intact butterflies, when light is on at 6am-6pm were tethered, see that they fly in south-westerly direction o Can describe behaviour by using tethered butterflies and looking at orientation  Orient in a certain direction  Time compensated o If the day begins at noon, monarch butterflies go NORTH  In the spring – go from Mexico back north  Significantly oriented flight but in the opposite direction o When removing antennae what happens to behaviour?  When flying without antennae, see that they fly in all sorts of direction  NO directive orientation flight  Lack description of migrative behaviour  Lose behaviour entirely o When the lights go on at 12 without antennae, also show disoriented flight  Antennal clock o Without antennae, behaviour disappears and is therefore necessary for behaviour o Control butterflies go south-easterly o Without antennae, that behaviour changes o Maybe, if taking antennae off, it might change how it goes to Mexico  Need to control chopping antennae off o if clock is functioning by perceiving the light of day, by blocking amount of light coming into antennae, maybe can change behaviour o Study: painted some antennae black with nail polish  Control: used CLEAR paint  Good control because it lets light in  Controls for action of painting antennae and light coming in  also blocks olfaction o results:  those painted with clear paint – orient in same direction of control  When given 6 hr delay, also orient properly (westerly)  When painted black, direction shifts entirely  Removing visual cues can change behaviour of monarch butterflies  Day starting at 6am – fly north  Da
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