Movement of genetically modified insects for research purposes

Summary Insects play a crucial role in research. Many laboratories are developing technologies to control insect vectors or agricultural pests by using genetic modifications that either reduce insect reproduction or increase refractoriness to disease transmission. Those tools include gene drive elements that may spread such genetic traits in a self-sustaining and cost-effective manner. Since international research collaborations are nowadays routine, movement of genetically modified insects between laboratories of different regulatory jurisdictions is very common. This article describes the requirements and guidelines for transportation of genetically modified insects for research and the experience gained by an Italian laboratory as a research centre involved in several shipments of wild and modified mosquitoes within an international research consortium aimed at developing sustainable tools for malaria control.


Introduction
It is difficult to overestimate the importance insects play in research.
41_1_08. Simoni -pre-print 2/15 Drosophila strains and mutants, is a very common practice between researchers, as reported by Cook   for the use and testing of genetically modified insects for population control (8) follows a step-wise approach for efficacy and safety tests at first in contained laboratories in regions where the target organisms are not endemic. As a consequence, movement of both wild and GM strains between different partner laboratories is necessary to perform initial regulatory studies, before importing the GM strains into contained use facilities in destination countries for further regulatory studies.
The Polo GGB exports both wild-type and GM strains to collaborators and to partner laboratories in endemic countries, subject to specific permits being obtained from the relevant national authorities and intellectual property agreements set by the research partners. This may include export of insects from wild-type strains that were originally collected in the field or GM mosquitoes based on those same background genotypes after they are approved to be tested in confined settings or eventually released in the field in the endemic country.

Documentation required for shipment
Shipment of live GM insects is subjected to international standards as well as national regulations that are specific for each country. The 41_1_08. Simoni -pre-print 5/15 documents required for shipping insects (e.g., mosquitoes) for research purposes distinguish whether the insects are considered an 'infectious substance' (or are infected and can transmit a pathogen). If the GM insect is also classed as an infectious substance, this has priority over it being genetically modified, and those more rigorous requirements should be followed. Insects carrying pathogenic agents are subjected to special rules and permits that will not be addressed in this article, as our experience is based on pathogen-free insects. Also, permits for deliberate release of GM insects into the environment are addressed by different regulations and are not discussed here. A shipment may be stopped by Customs officials due to incorrect or incomplete shipping paperwork, leading to delays in shipments that can compromise the survival of material and its usefulness in the destination country.
Shipping live mosquito eggs is always challenging and requires coordination between the sender, the logistics providers, the courier, and the recipient to ensure correct transport and timely arrival. Due to its complexity, it is suggested to rely on specialized logistics companies PI959 -shipping containment as Class 9). GMOs also need to be packaged to maintain any biosecurity containment requirements during ordinary handling in transportation. This requires a triple packaging system ( Fig.1): i) Primary receptacle, which is a leak proof sealing bag that contains the mosquito eggs; ii) secondary container, a leakproof large plastic tube that is used to enclose and protect the primary receptacle; and iii) rigid outer packaging, in which the secondary container is placed with suitable cushioning material. Outer packaging protects their contents from external effects, such as physical damage.
Additional precautions can be requested, for example adding a Parafilm or appropriate tape around the lid of the container to prevent any leakage. The material to be transported must be labelled in such a 41_1_08. Simoni -pre-print 9/15 manner as to notify any handler that the item to be transported contains GMOs. For this reason, the label UN3245 must be displayed on opposite sides of the outer packaging on a background of a contrasting colour and must be clearly visible and legible.
Each layer of packaging must be of such construction and sturdiness to independently prevent the release of the material under normal conditions during transport (temperature, humidity, pressure), and each layer must be independently closable or sealable.
It is recommended to ship mosquitoes as eggs or non-flying stages. This is because movement of reproductively viable stages of insects, especially to countries where pathogens of diseases are present and known to be vectored by such insects, might pose health risks.
Handling and shipment of non-motile life stages reduces the risk of escapees and also increases survival during transportation, since eggs are usually more resistant than adults. However, the non-motile eggs need to be protected from extreme heat, cold, and desiccation during transport, therefore insulated or adequate containers are necessary to maintain insect viability. If transportation is anticipated to be lengthy, the package may additionally include a cold pack to help regulate temperature. Most insects do not require large containers, feeding or added water during transportation (see the case described by Denton et al. [13], this issue, as an exception). However, depending on the species of mosquito (e.g. Anopheline), eggs might need to be kept wet for survival. Wrapping eggs in damp filter paper inside a sealed bag is usually sufficient and no free water should be used to minimize the risk of leaking.

Movement of insect carrying gene drives
There are no regulations specific to transport and movement of insects (or organisms, in general) carrying gene drive elements, and the documentation for gene drive organisms is not different from those for non-driving GM strains (14). However, recommendations and guidelines for movement of gene drive organisms are in place to ensure additional precautions are observed (5,6,7). Those

Conclusions
Exchange of live insects between institutions for research purposes is a very common practice, involving both wild-type and transgenic strains. When shipping mosquitoes carrying gene drive elements it has also been recommended to validate the strain using molecular procedures both before and after transport to ensure there are no unintended genotypes linked to the drive trait.
Regulations for import and transit of GM insects for research differ from country to country, and recommendations currently exist to guide the researcher to ensure proper transportation between laboratories. A 41_1_08. Simoni -pre-print 11/15 key factor is that these shipments are destined for laboratory studies, not field releases, which have different requirements and ramifications.

Glossary
Genetic modification: The process of altering the genetic composition of an organisms by use of genetic engineering in a way that does not occur naturally.
Gene drive: i) It is a process by which a genetic element (natural or synthetic) biases its own inheritance in respect to the rest of the genome, even in the presence of some fitness cost. ii) A gene drive is also the genetic elements that can cause the process of biased inheritance in its favour. iii) Gene drive refers also to the tool or technology to achieve a goal, for instance when referring to the use of gene drive mosquitoes to control a population.
Introgression (or introgressive hybridization): it is the process of