Orco Expression in Insect Testes

April 25th, 2014

Juan Carlos Malpartida

Why mosquito olfaction?

OR complexes and Orco

OR

co

- Heterodimeric ligand-gated ion channels. - Orco co-receptor + ORX tuning OR = functional

OR complex. - Tuning Ors bind to to narrow subsets of

odors/ligands. Requires Orco to function. - Orco is highly conserved among insects. Can

function without tuning ORs.

ORs found in Non-olfactory Tissues

-Insects: Ors expressed in An. gambiae proboscis, a gustatory appendage. - Humans: ORs expressed in reproductive tissue.

Spehr et.al. Science 2003

Evidence suggests Orco presence in An. gambiae spermatozoa

0

5

10

15

20

AgOrCo AgOr35 AgOr15 AgOr13 AgOr24

RP

KM

(b

od

ies)

R. Jason Pitts et.al. BMC Genomics 2011

- RNA Seq. show OR transcript levels elevated in male abdomen.

- RNA Seq. and RT-PCR show OR transcript in testes, low Orco transcript levels.

- Hypothesis: Orco protein present despite low transcript.

Pitts, Liu, Zhou, Malpartida, and Zwiebel (2014) PNAS

Immunohistochemistry Primary Antibodies: 1. Rabbit anti-Orco (IC3 conserved region of Orco) (1:500 dilution) 2. Mouse anti-α tubulin (1:500 dilution) Peptide Blocks: 1. Orco IC3 Peptide (11 a.a. long) (Specific Block) 2. Or18 Peptide (Non-specific Block)

Secondary Antibodies: 1. Goat anti-Rabbit (488 nm, Green) (1:250

dilution) 2. Goat anti-Mouse (647 nm, Blue) (1:250

dilution) Nuclear Stain: Propidium Iodide (nuclear stain, Red) (1:2000

dilution)

Example: An. gambiae testes (zoom) A = No block B = Non-specific block C = Specific block

IHC shows Orco Protein Presence

Pitts, Liu, Zhou, Malpartida, and Zwiebel (2014) PNAS

- Used antibody specific for IC3 conserved region of Orco. - IHC of An. gambiae (above) showed Orco expression in

spermatozoa.

What is the role of ORs in spermatozoa?

Pitts, Liu, Zhou, Malpartida, and Zwiebel (2014) PNAS

- Sperm behavioral assays conducted to see effect of Orco activation.

- Exposure to known ORco agonists caused increase in sperm flaggela beating.

- Could ORs be used in a reproductive context in An. gambiae?

- Population control protocols.

- Orco expression recently found in D. melanogaster and Ae. aegypti spermatozoa.

Are ORs part of insect reproductive biology?

• Is Orco present in the testes/sperm of other insects? Use IC3 Orco antibody to look for ORco sperm expression.

Finding Orco in insect testes:

Nassonia vitripennis,

Hymenoptera (Orco found in genome)

Aedes albopictus, Diptera (Orco found in genome)

Insect Phylogeny

IHC Cryosectioning Protocol

1. Individual testes collection and over night fixation with PFA.

2. Cryosectioning

3. Application of primary antibodies (1:500)

4. Application of secondary antibodies (1:250)

5. Visualization (Inverted Confocal Microscope)

Aedes albopictus

Ae. albopictus Testes Fragment, No Block (63x)

α-Orco Propidium Iodide

α- α-tubulin Composite

CS

α-Orco Propidium Iodide

Composite α- α-tubulin

Ae. albopictus Testes Fragment,

Non-Specific Block (63x)

α-Orco Propidium Iodide

Composite α- α-tubulin

CS

Ae. albopictus Testes,

Specific Block (20x)

α-Orco Propidium Iodide

α- α-tubulin Composite

CS

Nasonia vitripennis

Seminal Vesicle, Circle Shaped

Testis, Oval Shaped

Sperm-containing Organs in N. Vitripennis

Anterior

Posterior

Composite α- α-tubulin α-Orco Propidium Iodide

N. vitripennis Testes Fragment (40x)

No

Blo

ck

Spe

cifi

c B

lock

CS

α-Orco α- α-tubulin Propidium Iodide Composite

N. vitripennis Seminal Vesicle (40x)

No

Blo

ck

Spe

cifi

c B

lock

CS

Spring 2014: IHC Whole Mount Protocol

1. Collect last two segments of abdomen, containing testes, and fix for 3 hours (instead of overnight).

2. Apply primary antibodies (48 hours).

3. Apply secondary antibodies (overnight).

4. Mount whole tissue onto slide. 5. Visualization (Inverted Confocal

Microscope)

Mathias, Harvey, and Hogan (2004) Physiology

Whole Mount N. vitripennis Trials Conclusions from N. vitripennis WM trials:

• Bouin’s Aqueous yields a better signal than PFA (4%)

• 1 day old unmated male Nasonia yield bigger and less delicate samples than older mated.

• Optimum OrCo signaling is found using a dilution of 1:1000.

• Tubulin primary antibody does not affect Orco signaling; sufficient signal at 1:2000 dilution

• High propidium iodide masks Orco signaling.

• Vectashield + PI will give sufficient nuclear signaling.

Spermatogenesis in N. vitripennis

Clark, Bailey-Jourdain, Ferree, England, Sullivan, et al. (2008) Heredity.

Schematic of N. vitripennis spermatogenesis.

Elongated Spermatocysts.

N. vitripennis Whole Testes

No Peptide Block (20x) α-Orco

WM

A P

A

orco

B

C

overlay

No Block Non-Specific Block Specific Block

α-O

rco

C

om

po

site

WM

N. vitripennis Whole Testes

(20x)

A

P

A P

A

P

orco nuclear overlay

overlay

N. vitripennis WholeTestes 20x (above) 63x (below)

Orco signal in head/tails?

WM

A

P

α-tubulin orco

nuclear overlay

A D B C AR

SV

N. vitripennis Testes

Z-Stack Projection Magnification:40x

No peptide

Orco signal in sperm heads?

WM

A

Conclusions:

• Blockable Orco signal found in Ae. albopictus and N. vitripennis testis, possible Orco expression.

• Possible localization of Orco to sperm heads and/or tails of N. vitripennis spermatozoa.

• First steps towards novel population control protocols for disease vectors and pests.

Future Directions: • Sperm behavioral assays on

Ae. albopictus and N. vitripennis.

• RT-PCR, RNA Seq.: Transcript?

• Western blot: Correct protein size?

• Continue studies on cricket and cockroach testes.

Acknowledgements

- Dr. Willi Honegger

- Dr. Chao Liu

- Dr. RJ Pitts

- Dr. LJ Zwiebel

- Dr. Xiaofan Zhou

- Honors Committee Members

- The Zwiebel Lab

- The Bordenstein Lab

- The Page Lab