Collecting and Transporting *Helicoverpa* Samples Between Laboratories

Regulatory prerequisites

• Institutional biosafety approval and risk assessment on file.
• If samples are alive or cross state/national borders: obtain permits under the Biosecurity Act 2015 and relevant state biosecurity laws, and follow courier/IATA rules (incl. dry ice UN1845 marking).
• Confirm whether ethanol volumes are allowed by the carrier. Prefer RNAlater, freezing, or “no free liquid” shipping for air.
• Confirm receiving lab containment (PC2 insectary for live Helicoverpa armigera).

Roles

• Collector: Performs collection, labeling, primary preservation.
• Sender: Packages, completes manifest, dispatches.
• Receiver: Accepts shipment, verifies integrity, logs into LIMS, initiates quarantine procedures.

Safety

• Treat Helicoverpa as an agricultural pest. Prevent escape at all times.
• PPE: lab coat, gloves, eye protection.
• Decontaminate surfaces and tools with 70% ethanol or 0.5–1% bleach then water rinse.
• Waste: autoclave or freeze-kill then seal for disposal per local rules.

Materials

• Preprinted barcodes or waterproof labels; indelible marker.
• Forceps, paintbrush, aspirator, fine scissors.
• Leak-proof screw-cap tubes (2–15 mL) with O-rings; vented insect containers for live transport.
• Preservatives: 95–100% ethanol (DNA) or RNAlater (RNA).
• Absorbent tissue, zip bags, secondary watertight containers, rigid outer box/cooler.
• Cold packs; optional dry ice or liquid nitrogen dry shipper.
• Chain-of-custody (CoC) form and sample manifest.

Pre-trip preparation (Sender)

1. Exchange permits and approvals; confirm receiving address and responsible person.
2. Pre-assign Sample IDs and barcodes. Prepare manifest with fields in Section 7.
3. Calibrate GPS or note precise site/lab room coordinates where collected.
4. Stage clean collection area, dedicated tools, and labeled containers.

Collection at the collaborating lab (Collector)

General steps

1. Don PPE. Lay down clean bench-liner.
2. One sample = one unique ID. Label before transfer.
3. Avoid cross-contamination: change gloves and tools or flame/EtOH clean between samples.
4. Record metadata immediately (Section 7).

By objective

A. DNA (genomics)

• Small larvae/eggs: place whole in 95–100% EtOH at ≥1:10 tissue:ethanol.
• Larger larvae/pupae: cut 1–3 mm tissue tip; submerge in EtOH. Optionally change EtOH after 12–24 h.
• Keep cold but above freezing; no dry ice inside primary tube.

B. RNA (transcriptomics)

• Submerge in RNAlater per manufacturer volume; hold 4 °C overnight, then cold ship or freeze.
• For large larvae, dissect a small piece; avoid overfilling.

C. Live colony transfer

• Only with permits and receiving insectary.
• Place ≤10 larvae per ventilated container with untreated host leaves and a damp paper square. No soil. No free water.
• Label container “LIVE PEST INSECTS—DO NOT OPEN.”

D. Morphology

• Kill by freezing or brief EtOH immersion. Keep specimens straight; support with tissue.

E. Associated materials

• Host leaf with eggs: slide into vented container; avoid crushing.
• Frass/plant swabs: 2 mL tube, dry or in RNAlater depending on analysis.

Packaging for transport (Sender)

Triple containment (all preserved material)

1. Primary: sealed screw-cap tube; Parafilm the cap; add absorbent.
2. Secondary: watertight zip bag or secondary container with more absorbent.
3. Outer: rigid box or cooler with cushioning. Include printed manifest outside the secondary but inside the outer.

Temperature

• DNA in EtOH: cold packs OK.
• RNA in RNAlater: cold packs or frozen packs; dry ice allowed if compliant with UN1845 marking and net weight on the box.
• Frozen without liquid: ship on dry ice or in a dry shipper.
• Live: room-temp with cool pack buffer if hot. Never chill below ~8 °C for larvae.

Labels and documents

• Outer box: sender/receiver, emergency phone, “Biological Samples—Non-hazardous, Non-infectious,” temperature instructions, and dry ice label if used.
• Include permits and CoC copy.

Dispatch

• Use overnight service. Avoid weekend holds. Share tracking with Receiver.

Receipt and quarantine (Receiver)

1. Inspect outer box for damage and correct labels. Photograph if damaged.
2. In a containment area: open secondary, verify no leaks/escapes.
3. Reconcile items with manifest. Sign CoC.
4. Live samples: move immediately to PC2 insectary/quarantine cage. Record survival and condition.
5. Preserved samples: log temperatures and condition; move to −20 °C (DNA) or −80 °C (RNA) storage. Replace EtOH with fresh if cloudy.
6. Decontaminate packaging surfaces and dispose per waste plan.
7. Enter all records into LIMS and assign storage locations.

Metadata to record (minimum)

Sample ID, taxonomy (Helicoverpa sp., life stage, instar if known), condition (live/frozen/EtOH/RNAlater), host plant species/part, collector, source lab, date/time, room/location (GPS or building/room), rearing or pesticide exposure history, preservation type and volume, temperature at sealing and at receipt, permits/approval numbers, intended analyses. Optional: photo, mass/length, notes.

Chain-of-custody (CoC) and manifest

CoC line item

• Sample ID → Sender name/sign/date/time → Courier tracking → Receiver name/sign/date/time → Condition notes.

Manifest columns Sample ID | Taxon | Life stage | Preservation | Quantity | Container type | Temp requirement | Source lab/room | Host plant | Notes | Permit IDs.

Waste and decontamination

• Plant material, frass, disposable tools: autoclave or freeze-kill then sealed disposal.
• EtOH waste: flammable-liquid container for chemical disposal.
• Surfaces and reusable tools: 70% EtOH or 0.5–1% bleach then water rinse; dry.

Records and retention

• Keep permits, manifests, CoC, temperature logs, and incident reports ≥5 years or per institutional policy.
• Version this SOP; review annually.

Notes

• Regulations vary by jurisdiction and carrier. Verify permits and packaging rules before each transfer.
• When in doubt about ethanol in air cargo, fix specimens, decant to minimal residual liquid, and ship cold or frozen, or use RNAlater.