Lab III – Aiptasia: Genotyping and Symbiosis Notes (Page-by-Page)

Objective: Genotype individual Aiptasia without sacrificing the animal. Animals will be bisected; one half for DNA extraction and genotyping using SCAR markers, the other half will grow into a clone with the sacrificed half's genotype.
Lab objective: Learn about corals, symbiosis, and the Aiptasia model.
Background (essential points):
- Coral reefs are a key, productive ecosystem with high biodiversity and multiple ecosystem services.
- Reefs occupy a small fraction of the planet but support large marine life and human industries (e.g., tourism, fisheries).
- Global threats include pollution, acidification, and warming; global decline around $50\%$ over the last $30$ years; projected survival to $2050$ is roughly $10\%$ of historic populations.
- Symbiosis: corals host photosynthetic dinoflagellates ( Symbiodinium ) that provide up to $90\%$ of coral energy; breakdown leads to coral bleaching when algae are expelled.
- GFP/fluorescence beacons: corals’ fluorescent proteins may help recruit symbionts; researchers study GFP expression to understand symbiosis and bleaching recovery.
- Aiptasia ( Exaiptasia diaphana ) as a model: ubiquitously cultured, aposymbiotic capability, easy spawning, and a useful proxy for coral symbiosis studies.
Model context: Two worldwide strains exist in the lab context (CC7 and H2); Aiptasia can be used to explore molecular mechanisms of symbiosis before working with hard corals.

Corals are colonial cnidarians in the class Anthozoa; sea anemones are the closest relatives of reef-building Scleractinia.
Key biology:
- Hard corals secrete calcium carbonate exoskeleton; colonies are composed of many polyps.
- Symbiodinium live inside coral tissue and photosynthesize, supplying energy to the host.
- Mutualism: algae receive protection and substrates; corals receive photosynthetic products (up to $90\%$ of energy).
- When stressed, symbiosis breaks down → algal expulsion (bleaching); bleached corals may die if symbionts are not re-recruited.
Global trend and rationale for study:
- Bleaching events contribute to reef decline; understanding molecular/genetic controls of symbiosis may aid conservation.
- Researchers use reverse genetics (gene expression manipulation) to probe symbiosis mechanisms.
GFP/beacon concept:
- Some corals produce GFP-like proteins; experiments suggest GFP-related signals may influence symbiont recruitment.
Aiptasia model specifics:
- Aiptasia allows induction of symbiotic and aposymbiotic states for studying dissymbiosis and symbiosis dynamics.
Strains in the lab:
- CC7 (Caribbean) and H2 (Pacific); two CC7 tanks (Endo, CB) and one H2 tank.
Culture conditions:
- Artificial seawater (ASW), salinity $33-35\ \text{ppt}$ , pH $8.0$ .
Genotyping plan:
- Use SCAR markers to distinguish strains sans harming the animals.

Laboratory setup basics:
- Two global strains (CC7 and H2); CC7 tanks named Endo and CB; one H2 tank observed.
- Animals kept in ASW at $33-35\ \text{ppt}$ , pH $8.0$ .
Genotyping goal:
- Apply SCAR markers to rapidly identify strain/genetic background in living specimens.
Housing note:
- Stock tanks and routine maintenance located in the Lab Fish Room; Figure references show stock layout.

Basic Anatomy (Cnidaria):
- Polyp organization: radially symmetric, sac-like body, two primary cell layers (gastrodermis and epidermis) separated by a gelatinous mesoglea (hydrostatic skeleton).
- Primary tissues: endoderm becomes gastroderm; ectoderm becomes epidermis; mesoglea provides structural support.
- Body plan: a column and oral disk with a mouth; tentacles surround the mouth; cnidocytes with nematocysts for prey capture.
Coral vs. anemone anatomy:
- Corals: polyps sit in corallites with calyx; connected via coenosarc; secreting exoskeleton (calcium carbonate).
- Aiptasia: solitary anemone; lacks exoskeleton; adheres to substrate via basal disk.
Shared features:
- Both have cnidocytes and nematocysts in the tentacles; endoderm houses Symbiodinium symbionts in the tissue.
- Both possess a dispersed nerve net and a muscle layer around the body column for movement.
In-lab activity (labeling):
- In ASW, observe and label: tentacles, mouth, mesoglea, basal plate, gastrodermis, epidermis, gastrovascular cavity, oral disc.

Feeding biology:
- Cnidarians are predatory and rely on nematocysts to capture prey; although symbiotic, they still feed on small zooplankton.
Feeding protocol in lab:
- Feed with newly hatched Artemia (brine shrimp) nauplii: harvest from hatched eggs, add to dishes, observe prey capture.
Observation task:
- Describe the anemone’s response to feeding (watch under dissecting scope).
Figure reference: Artemia life cycle (Fig. 4).

Hydrostatic skeleton and movement and defense:
- Aiptasia uses a hydrostatic skeleton with body-wall muscles to move; responses during handling are observable and rapid.
Key defensive structures:
- Acontia: threadlike, cnidocyte-rich tissues at the trunk base deployed when threatened.
Experimental observations (data collection):
- A) Observe response to fingertip proximity; infer “seeing” or sensing.
- B) Remove ASW; observe movement ability without a fluid medium.
- C) Poke trunk with a pipette tip; observe reaction.
- D) Poke repeatedly until acontia deployment; count pokes until response.
- E) Time-based monitoring: start stopwatch and check periodically; capture time to acontia retraction.
Data notes:
- Take time-stamped pictures to document responses.

Reproduction (Part IV):
- Cnidarians reproduce sexually (gametes from gonadal tissue) and asexually (budding or pedal disk).
- Aiptasia buds form via pedal lacerates from the pedal disk, creating genetic clones.
- Long-term goal: induce spawning in lab to study fertilization and genetic manipulation (knock-out/knock-in approaches).
Aims for this lab activity:
- A student test: smear an Aiptasia to see if many babies form; note strain.
- Procedure: obtain a small animal, place in clean ASW on a slide, chop/smash with a razor, transfer to clean ASW dish, observe next week for babies.
- Document with before/after images.
Reference: Figure 5 shows sexual vs. asexual reproduction in cnidarians.

Continuation of reproduction activity (Aiptasia smear test):
- Label the strain used for the smear; track which family/strain yields offspring.
- Keep slides in ASW in the fish room for next week’s check.
- Collect before/after images to record results.
Note: This is an exploratory, student-driven test to probe clonal propagation from tissue.

Post-smear handling and quadrant cutting:
- After recovery from ejecting condensations (acontia), cut the anemone into halves or quarters (as feasible).
- Store these fragments in dishes for future labs; properly label plates.
- Goal: preserve living tissue for continued use and potential clonal lines.
Practical note:
- Successful survival is not guaranteed; plan for disposal if necessary and maintain accurate labeling for traceability.