The Quarantine Laboratory: An Overview

Discovery and importation of new natural enemies is the key strategy in classical applied biological control programs. This is illustrated in Figure I which depicts basic implementation procedures for biological control projects. Most often, importation of newly discovered natural enemy species requires that host insect and plant material, along with unknown and/or possibly undesirable species, accompany the sought organisms. Therefore, biological safety is of primary and immediate concern to researchers working in biological control of arthropods. The main method of assuring safety during the importation of natural enemies is physical containment. This is achieved in the Quarantine Laboratory.

The Quarantine Laboratory is under the direction of the Quarantine Supervisor and the Quarantine Officer. These persons develop processing procedures, control access, and oversee the day-to-day operation of quarantine. They also serve as liaison with regulatory agencies, institutions and project leaders. Further, they participate in collaborative biological research, help coordinate natural enemy identifications with appropriate taxonomists, compile databases of quarantine activities, and consign natural enemies to projects following agreement with taxonomists and project leaders.

There are certain procedures that must be followed prior to importation of exotic material. In every case where living plant pests, some of which harbor parasitic natural enemies, are to be included in importations, USDA-APHIS-PPQ permits must be acquired through application utilizing PPQ Form 526 (Attachment 1). State approval is also required. The acquisition of importation permits is possible because required safeguards for issuance of permits (Safeguard Guidelines For Containment Of Plant Pests Under Permit, USDA-APHIS-PPQ) are met by the physical design of the Quarantine Laboratory (Attachment 7). It is thus obvious that biological control programs simply cannot proceed without a strong commitment to quarantine requirements, personnel and activities.

The physical containment aspect of quarantine is only a part of the safety criteria. There are specific regulations for persons working in quarantine that are as important as the containment laboratory. Criteria for the conduct of research in quarantine are written for individual laboratories, and are dependent on the structure of the laboratory and the nature of projects. For the most part, these criteria describe Quarantine Laboratory protocol for the following: entry of authorized personnel, receipt of exotic material, assigned working space, interior containment (eg. cages), organism handling procedures, waste disposal procedures, identification of natural enemies, required biological studies, consignment of natural enemies, egress, and information storage and retrieval.

Entry to the Quarantine Laboratory is limited to persons trained in quarantine procedure and methodology. These authorized quarantine personnel are generally Quarantine Supervisors, Quarantine Officers (and their staff), project leaders (whose names appear on approved PPQ 526 applications) and a limited number of assistant(s). Entry is strictly limited and all access to quarantine is controlled by the Quarantine Supervisor or Officer. The entryway is always kept locked.

Receipt of exotic material is the first step in the introduction of new organisms. There is a designated package opening area (room or space) in the Quarantine Laboratory where processing of incoming shipments begins. The opening area is generally equipped with a light trap, large sleeve cages and a convection oven or autoclave. Packages are opened inside large sleeve cages to minimize any risk of escape. All excess debris and packing material are cooked to kill unwanted organisms, and only the materials necessary to create natural enemy cultures, and to conduct taxonomic and biological studies, are allowed to leave the opening area. This immediate isolation eliminates much of the source of unwanted organisms.

Assigned working space further minimizes - or eliminates - the potential for contamination of isolated specimens and/or cultures. The work of authorized quarantine personnel is restricted to specific areas to further reduce the potential for contamination. In conjunction with specified working areas, suitable interior containment, such as sleeve cages, escape proof vials and closed humidity control systems, affords even greater security.

Handling procedures that isolate specific organisms in time and space complement the proceeding and further allay possibilities of contamination and inadvertent movement of organisms. These procedures include handling of host insect and host media prior to working with natural enemy cultures, scheduling natural enemy culture maintenance, and handling quarantined host (pest) organisms following all other procedures. This prevents the transfer of such a pest to a closely related host culture where it could go unnoticed.

Waste disposal procedures in the Quarantine Laboratory are designed to eliminate the inadvertent movement of living organisms to the environment. This is generally accomplished by trapping all liquid wastes in chlorinated or heated holding tanks prior to discharge into sewer systems; cooking or autoclaving solid wastes inside the laboratory; and incinerating all solid wastes when they are removed from quarantine. Air is cleansed through a series of screens and/or hepa filters.

Identification of natural enemies is one of the most important aspects of quarantine; the others being physical containment, operational criteria, biological studies and permanent records. The importance of systematics in biological control programs cannot be overstated. The initial taxonomic placement of natural enemy species provides the key to all the resources of the literature which often includes biological, ecological and historical information crucial to understanding the two major evaluative criteria for later consignment of natural enemy species from quarantine: host insect association and reproductive mode.

Biological studies are conducted in the Quarantine Laboratory to determine host associations (e.g. reproduction on target pest) and reproductive modes of natural enemies, particularly those with unknown or poorly understood systematics. The manner that parasitic Hymenoptera and Diptera reproduce on specific hosts is the single-most important biological characteristic used for evaluation of their suitability for consignment from quarantine. For example, parasites that reproduce on pure cultures (not previously parasitized) of specific hosts are primary parasites and are suitable for consignment. Parasites that reproduce females only on previously parasitized hosts are obligatory hyperparasites and are not suitable for consignment. Parasites that reproduce males on previously parasitized hosts and females on pure cultures of hosts are autoparasitic (or adelphoparasitic) and are suitable for consignment.

Primary parasites may also reproduce on previously parasitized hosts (multiple parasitism, facultative hyperparasitization), particularly under laboratory conditions. This capability may enhance survivorship but does not necessarily confer competitive superiority in multiple parasite systems. Differing searching abilities at variable pest population densities will modify the impact of this phenomenon. Such parasites are eligible for consignment following agreement by quarantine personnel, taxonomists and project leaders.

Consignment of natural enemies from the Quarantine Laboratory is the final assurance that specific organisms leave the containment area and others do not. Consignment is based on systematics, reproductive modes and host associations; all of which are established in quarantine on a case-by-case basis through interaction between the Quarantine Supervisor or Officer, the appropriate taxonomists, the project leader and other authorized quarantine personnel. Authorization to initially colonize exotic natural enemy species is gained through application to state agencies (departments of agriculture) and then to APHIS-PPQ by submission of taxonomic and biological information that documents natural enemies are not plant pests.

Federal regulatory processes relating to field colonization of non-plant-feeding arthropod natural enemies are being reformulated. Regulatory and statutory authority and regulated organisms are not clearly defined. It is imperative that initial field colonization of new natural enemies be coordinated with appropriate local and state agencies, and that researchers are active participants in the development of any new guidelines and regulations that affect the timely utilization of natural enemies.

Egress of personnel and material from the Quarantine Laboratory must also be done in a prescribed manner. Persons working in quarantine must wear laboratory coats that are used only for specific projects. These coats must be both donned and removed in the inner-most quarantine entry chamber. A light trap is always in operation in this chamber. Otherwise, the chamber is kept dark. Authorized quarantine personnel must cleanse themselves and any material that leaves the containment laboratory. Material that has come into contact with exotic plant material must be cleansed in sodium hypochlorite solution or alcohol (70-95% ETOH) before leaving quarantine. The Quarantine Laboratory must always be kept meticulously clean; organic trash should be held cold to eliminate secondary pests (eg. mites, mold) and all organic material (eg. media) must be held in closed containers.

Information storage and retrieval are vital aspects of the safe and timely operation of the Quarantine Laboratory. Because systematic and biological information are such important factors in biological control programs, the information generated in quarantine must be accurately recorded. This information can be utilized for reporting to regulatory agencies and for acquisition of historic data, such as association with voucher specimens deposited in various museums and collections.

The Quarantine Laboratory is the key link in the chain of events of applied biological control programs (eg. systematics, foreign exploration, importation, colonization and evaluation of natural enemies). It provides both the means to obtain the required importation permits, and the containment facilities necessary to receive exotic material and conduct initial taxonomic and biological studies on new natural enemies that are needed to solve key pest problems.

Statement on Quarantine Procedure, Natural Enemy Field Release Authorization, and Possible Categorical Exclusion from the Federal Plant Pest Regulations, 330.200, for Non-Plant-Feeding Natural Enemies of Arthropod Pests.

This is written to clarify our (biological control researchers at state institutions) concept of the operation of the Quarantine Laboratory for importation of arthropod natural enemies, and subsequent field release authorizations for non-plant-feeding arthropod natural enemies for utilization in programs for biological control of arthropod pests.

We import all exotic plant pest material under authorization of approved USDA-APHIS-PPQ Form 526 applications. Exotic material is processed in our institutional quarantine laboratories, which are fully approved for importation of exotic material by state and federal regulatory agencies. The laboratories are under the direction of Quarantine Supervisors and Quarantine Officers who control all access to the containment areas and all consignment of natural enemy species to project leaders.

First and foremost, we concentrate our initial efforts in quarantine on isolation of natural enemy species from plant feeders, with the intent to create pure cultures of single species of non-plant-feeding natural enemies on pure cultures of various arthropod hosts. In doing so, we eliminate the original plant pest species from specimens of non-plant-feeding natural enemies that are candidates for consignment from quarantine.

We consign such natural enemies to project leaders on a case-by-case basis. Suitability for consignment from quarantine is based on taxonomic identity of the natural enemy species, its host association(s) and its mode of reproduction.

Great emphasis is given to systematics in the entire quarantine procedure. Accurate taxonomic diagnosis of each natural enemy species is the only means to access pertinent literature. Documented biological, ecological and historical information is the most sure and efficient means to initially evaluate the status of natural enemies. Further, series of voucher specimens taken from parent and/or F1 material are curated and deposited in permanent collections for reference and study.

Following accurate taxonomic determinations, necessary biological and host association information is gained by research. For the parasitic natural enemies, our concern is the reproductive mode of each species. This includes determination of primary parasitization, facultative hyperparasitization or obligatory hyperparasitization. Determination of the reproductive mode is accomplished at the same time that arthropod host association is tested. Parasitic and predatory natural enemy species of arthropods that are biologically suitable for consignment are not plant pests.

Natural enemies consigned from quarantine are therefore suitable for field release. Otherwise, the species are destroyed in the Quarantine Laboratory. Request for authorization for initial field release is the responsibility of the project leader. Authorization is sought by written application to the appropriate state (and county, if required) department of agriculture. When such applications are approved, the states may seek approval from USDA-APHIS-PPQ. This has been accomplished without undue delay, and can now be done more rapidly, thanks to electronic mail and FAX. Again - for emphasis - these requests for approval are for non-plant-feeding natural enemies that have been completely isolated from original plant pests.

Proposed categorical exclusion of certain non-plant-feeding natural enemy groups from the Federal Plant Pest Regulations, 330.200, is intended to aid USDA-APHIS-PPQ in initial field release authorization decision making. Such a list of excluded natural enemies has been put forward by ARS, aided by state researchers, but is not complete or operational.

Regardless of categorical exclusions, we will continue to conduct our quarantine evaluations as previously described; on a species-by-species basis. We will continue to rely on systematic and biological information to determine the suitability of natural enemies for consignment from quarantine. We will continue to consign only those non-plant-feeding natural enemies that are biologically suitable for field release programs, and that have been completely isolated from any original plant/pest association. We regard case-by-case studies of newly discovered species in quarantine as the only sound procedure to determine the suitability of natural enemies for consignment.

In order to assist USDA/APHIS/PPQ in their decision making process through environmental assessments that specify non-plant-feeding arthropod natural enemies are not plant pests and are suitable for field release, we will continue to provide accurate systematic and biological information to state and federal agencies for initial colonization of species new to the U.S. Further, we have attached here (Table I) citations with lists of natural enemies that have been previously utilized in programs for biological control of arthropod pests. These lists contain examples of the kinds of organisms that are suitable for field release programs.

Table I. Literature

Introductory Summary

The Biological Control Quarantine Laboratory at Texas A&M is utilized for importation and containment of arthropod pests and their arthropod natural enemies. USDA-APHIS-PPQ permits (PPQ Form 526) are obtained prior to importation and introduction of organisms under quarantine stricture.

All field material imported under PPQ permit is initially processed in the Quarantine Laboratory. Taxonomic status of natural enemies is determined prior to consignment of live specimens from the Quarantine Laboratory. Reproductive modes of unknown, unidentifiable, or problematic species are determined in the Quarantine Laboratory.

Only the Quarantine Supervisor permits use of and entry to the Quarantine Laboratory. Project leaders and authorized quarantine personnel are permitted entry to assigned areas of the Quarantine Laboratory for specific time periods on a case-by-case basis.

The Quarantine Laboratory was designed specifically to meet all of USDA-APHIS-PPQ safety criteria as stated in Safeguard Guidelines for Containment of Plant Pests Under Permit. A copy of these safeguards follows immediately, along with comments pertinent to the A&M laboratory. The following safeguards have also been approved by officials of the Texas Department of Agriculture.

Conditions of Safeguard

Function, Operational Format, Procedure and Protocol

The Quarantine Laboratory was designed and is intended to operate as an entity within the Biological Control Laboratory. The purpose of the Quarantine Laboratory is to safely facilitate the introduction and study of exotic natural enemies of insect and arachnid pest species for use in biological control programs, and to engender international exchange of natural enemies. Further, introduction of exotic material through the Quarantine Laboratory provides the means to construct permanent reference collections of natural enemies and their specific hosts for taxonomic study.

All exotic material introduced through the Quarantine Laboratory, other than pure cultures of predators and primary parasites, and some required host material, must do so under USDA-APHIS-PPQ permit as authorized by PPQ Form 526. Prior to introductions, a fully authorized copy of this permit must be given to the Quarantine Supervisor (Q.S.). All use of the Quarantine Laboratory requires approval by the Q.S.

The Quarantine Laboratory is not a means of introducing curiosities unrelated to biological control programs.

Responsibilities of the Quarantine Supervisor

The Q.S. is responsible for the supervision and operational function of the Quarantine Laboratory. These responsibilities include:

Responsibilities of the Project Leaders and Authorized Quarantine Personnel

Entry to the Quarantine Laboratory

Only the Q.S. has authority to approve entry to the Quarantine Laboratory. Project leaders may acquire approval through discussion or correspondence with the Q.S. A list of Persons Authorized to Enter Quarantine will be posted on the outside door of the Quarantine Laboratory. Authorization to enter shall be for a designated time period that will be noted on the approval list.

P.L.'s may also request that a member(s) of their staff be approved for entry (A.Q.P.). Again this authorization may be acquired through discussion or correspondence with the Q.S.

Those "Persons Authorized To Enter Quarantine" may do so only when approved through discussion or correspondence with the Q.S. The following protocol must be observed.

Regulations and Procedures


There is an inter-com system adjacent to the entry and exit doors. If you are uncertain about entry or have pertinent questions, use the inter-com. Don't be demanding about inter-com or telephone use. It may not be possible to answer because of on-going procedures.

Inside the Quarantine Laboratory

Upon entry go to the assigned work area designated for use by a specific project. Designated work areas shall be assigned through correspondence with the Q.S. and are temporary. Methodology, means of containment and duration of entry authorization will be decided by correspondence with the Q.S. when space is assigned.

The Quarantine Laboratory is, by its very nature, intended to be a quiet and clean area. All trash must be held in closed containers. Ziplock bags are very suitable for small refuse. Large, closed containers will be available for other disposal. Clutter and accumulated debris are not acceptable and will be destroyed. It is the responsibility of each project to police their assigned work space prior to egress from Quarantine.

Knock prior to entry of any door. Wait for a possible reply, and enter when there is a positive response, or no response.

When work is completed on a given importation the assigned area must be completely cleaned and made ready for further use.

Records, Identifications and Consignment

All shipments are assigned a unique code. For example, T89001 represents Texas A&M Quarantine Laboratory, 1989, shipment number one. In order to utilize an information storage and retrieval system this number must always remain associated with original material, voucher specimens, and cultures (which may later be combined).

A.Q.P. should recognize that this code can be used to separate collections. For example, collections made from different host plants or geographic regions should be assigned different codes initially. Further, rather than placing 2 or more species (or populations) received from laboratory culture under one code, it is preferable to assign each organism a separate code. The addition of code integers (i.e. T89001-1) at a later date will not fit our information system.

Sender/Receiver (S+R) Forms(Attachment 3) are to be utilized to provide the information necessary to assign unique numbers and to complete the collection data areas on identification forms. The sender must complete the card accurately and legibly. S+R cards are available from the Secretary for the Biological Control Laboratory.

In every case, the form Identification Of Material Imported Through Quarantine (Attachment 4) must be completed for each natural enemy species that is pertinent to the project. Curated specimens and the identification form must be given to the appropriate taxonomist(s). Again, only determinations on signatured identification forms are complete and only these will be entered in the Quarantine database.

Original, signed forms are returned to the Q.S. and become part of the permanent record. Project personnel and taxonomists may make copies of the completed identification form for their files before returning the original to the Q.S.

It is not necessary to complete a new form each time live material is consigned after initial identification of a given organism. However, the form should always be associated with dead original material, following recovery after exposure to hosts in the laboratory, for example, when passed to a taxonomist for identification. These forms are permanent records and constitute the basis of the Quarantine database. Forms are available from the Q.S. and the Secretary of the Biological Control Laboratory.

Unidentified material cannot be consigned to leave the Quarantine Laboratory alive. As a major role of the Quarantine Laboratory is to consign natural enemies to the P.L. - and thus for colonization in the field where they can become useful - there must be a great deal of emphasis given to providing the taxonomists with material that can best be utilized to make rapid identifications.

Dead original material should be kept dry and curated as per a taxonomist's instruction. Live Ichneumonoidea should be killed in a killing jar and point-mounted. Live Chalcidoidea (except Aphelinidae, see following) should be killed directly in 70% ethanol in clean homeopathic vials and closed with green neoprene stoppers2. The ethanol should completely fill the vial. Curation of Chalcidoidea should be discussed with J. B. Woolley on a case by case basis. All original material and (their) progeny must be identified by the importation code number and all collection data must be provided to the taxonomist on labels and on the Identification of Material Imported Through Quarantine form.

Aphelinidae should always be preserved dry; preferably between layers of clean cotton. Do not point-mount Aphelinidae.

Generally, the following persons will examine and provide taxonomic determinations of natural enemies:
A major effort is underway to compile a permanent voucher specimen collection in the TAMU Entomology Collection. Color-coded labels are utilized to identify and enhance rapid search for voucher specimens. The completed form Information Required For Submission Of Vouchers (Attachment 5) must accompany each species submitted for housing in the TAMU Collection. These forms are available from the taxonomists and the Q.S.

Project personnel should arrange for taxonomists to look at live material by discussion with the Q.S. and taxonomists on a case-by-case basis.

It is not expected that taxonomists can always determine the taxonomic status of natural enemies from examination of living specimens. However, in many cases the taxonomist can place live natural enemies in genera or groups. In such cases the Q.S. will discuss consignment with the taxonomist and the P.L. Following the death of such natural enemies, the consigned original material must be properly curated and returned to a taxonomist(s) for identification as just previously described.

The P.L. and his staff have oftentimes gained considerable experience and familiarity with natural enemy groups associated with pest species which are the object of specific research projects. Often the P.L. can rapidly determine the taxonomic status of imported natural enemies based on this experience and familiarity. Consignment in such cases will also be through agreement with the Q.S., taxonomist(s) and P.L.

No live organisms may leave the Quarantine Laboratory unless authorized by the Q.S.. Authorization of consignment is shown on the identification form. It is the responsibility of project personnel working in Quarantine to create a daily record of all consigned live organisms prior to their leaving the Quarantine Laboratory. Each importation will have separate files identified by code number, name of project leader and country of origin. Consignment records are to be kept in these files. All specimens must leave the Quarantine Laboratory in clean containers.

The P.L. can do a great deal to hasten obtaining natural enemies from the Quarantine Laboratory by providing advance information to the Q.S. and the taxonomists by utilization of the form Anticipated Arrival Of Shipment Under PPQ Permit.

A flow diagram (Attachment 6) summarizes the basic steps taken to manage a typical importation.

Egress from the Quarantine Laboratory

Space Allocation and Use

In essentially every case working space must be shared. The major concerns with shared space are: The general lack of isolated working space requires that space be designated for temporary use; taxonomic determinations and consignments be made rapidly; space be always meticulously clean; and every care and precaution be taken to avoid contamination of rearing units and the Quarantine Laboratory.

Each importation presents different and wide-ranging variables. Therefore, handling procedures for each importation can be different and require modification to suit current space availability and requirements, the nature of the organisms involved, and the general cleanliness of the incoming material. These matters can be more readily and astutely organized when Anticipated Arrival Of Shipment Under PPQ Permit forms are utilized.

The Q.S. will assign designated space. Special requirements (e.g. temperature, photoperiod) should be organized with the Q.S. when the Anticipated Arrival form is completed.

Summary

Accurate taxonomic determinations are a major aspect of biological control projects. The form Identification Of Material Imported Through Quarantine provides a permanent record of natural enemy species identifications and specific host associations. This form is the information source for the Quarantine database.

The forms attached to this document are not intended to replace communication; they are to become part of the Quarantine record and are intended to enhance the success of the Biological Control Laboratory and the persons working therein.

Quarantine regulations are best fulfilled with a sense of care. If each person working in the Quarantine Laboratory takes the time to reflect on the reason and need for care, a sense of order will evolve that will allow the Quarantine Laboratory to function in an unobtrusive and positive manner.

All persons working in the Quarantine Laboratory are expected to comply with the standards and procedures that have been presented in this booklet. Questions should be directed to the Q.S.

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Last modified: April 16, 1996 by J. A. Jackman