Biosafety measures for wild-type stock

Plodia pantry moths are non-infectious, non-parasitic, non-vector, and non-exotic insects that are found on all continents, and generally considered as native pests of stored food products. They are notorious for their infestation potential in kitchens and pantries, and can proliferate within a laboratory building if baseline containment measures are not taken. We prescribe important recommendations for working with this insect at the wild-type state.

• Transfers between primary containers. The entire lifecycle is maintained in escape-proof containers, so the risk of egg/diet spills or flying moth escapes is limited to moments where these containers are opened. The transfer of narcosed adults to an oviposition jar should be quick, over a tray, and under a fumehood. Lab surfaces must be wiped with ethanol if potentially contaminated with eggs. The transfer of adults isolated in cups or flasks must be done inside a secondary container, such as a mesh cage.
• Secondary containment. Plodia cultures should be in a dedicated incubator or in a clean insectary with limited access and no direct escape routes. “Pantry moth” pheromone traps are highly effective for detecting an early infestation, and may help to catch accidentally released males before they mate. Indoor ultraviolet insect traps can also be installed as preventive measures in the insectary and adjacent rooms.
• Lab environment. Food must be prohibited in the laboratory areas surrounding the insectary. In addition, Plodia can infest the wax of laboratory bee cultures.
• Disposal and cleaning. Stringent measures to devitalize all the material for disposal in order to avoid a laboratory infestation. All containers, individuals (including excess eggs), cultures and waste (wipes) are sealed in a plastic bag and frozen at −20°C for at least 72 h in order to achieve complete disinfestation, a conservative procedure based on published measures of an LT95 estimate of 7 h at this temperature (Johnson, 2007). Dilute bleach (5%) treatment for 10 min dissolves egg chorions and is an effective chemical decontaminant for surfaces and equipment that can not be frozen.
• Personal protective equipment. Disposable gloves are recommended to avoid contaminating cultures with mold. Lab coats are recommended to avoid the export of egg material outside of the lab. Most importantly, experimenters must use respiratory protection (fumehood, respiratory filter) when opening or cleaning mass-rearing containers, due to the allergen potential of moth scale volatiles (Binder et al., 2001).

Arthropod containment level 2 measures for genetically modified stock

The generation and maintenance of transgenic or genome edited arthropod stock is prone to formal regulations that are country or institution specific. Published guidelines about Arthropod Containment Levels 1 and 2 (ACL-1 and ACL-2) protocols (American Committee of Medical Entomology-American Society of Tropical Medicine and Hygiene, 2019) provide an advisory framework for safe genetic modification in Plodia. While the ACL-2 standard was designed for arthropods of public health concern, it is flexible and overall a good target for responsible research conduct, but may be insufficient if the laboratory modifications present a risk of fitness advantage or genetic contamination in the wild (e.g. insecticide resistance, gene drives). In addition to the aforementioned baseline recommendations with wild-type Plodia stock, we advise the following when working with genetically modified Plodia.

• Disposal. All material must be treated as Biohazardous waste (similar to transgenic Drosophila) following ACL-2 protocols.
• Facilities. Simple ACL-2 barriers to flying and crawling insects, for instance using a dedicated insectary or incubator room with self-closing doors, and custom ventilation meshes, should be accommodated whenever possible. More expensive facilities with double-door vestibules, or negative air pressure, are not needed for Plodia, since accidentally released adult moths should be extremely rare with the aforementioned primary containment procedures.
• Personel. ACL-2 measures have increased stringency on access and training compared to ACL-1, which are important to reduce the risk of accidental releases due to negligence.