To develop an authentication matrix, a survey of all strains for known, available distinguishing characteristics is done initially. Considering the MR4 holdings, many strains are morphologically distinct from all others maintained such as An. dirus, An. minimus, or An. freeborni. For those, authentication standards were prepared based upon adult morphology. The remaining strains were sorted by species (i.e. An. gambiae s.s. - henceforth referred to as An. gambiae) and their known (as observed over time in our laboratory) genotypic/phenotypic traits were compiled. As an example, if the red-stripe character was noted in one strain, the remaining strains were surveyed for that character.

To create a matrix, the authentication methods initially identified were ordered from least to greatest in terms of cost and/or time consumption (see Table 1). Once the techniques applied generated a unique resulting combination, it was then possible to distinguish a cohort from all others available in the repository and to consider authentication complete for that strain. The objective of any authentication should be confidence to the level of the individual, rather than cohort, when possible. Methods were then analyzed to see where assays could be combined to save time and resources, aiming to complete authentication in the least number of steps (or in the least amount of time/cost) with a high degree of confidence.

Once the matrix is completed, each line of the table (see Table 1) represents an authentication method for a particular strain. Additionally, a flow chart was created that represents how an unknown cohort of An. gambiae reared at the MR4 can be distinguished (Figure 1). This decision tree presents a different way of thinking about the same authentication scheme. Of note, authentications must be conducted on a cohort, see discussion.

Phenotypic markers are highly useful in routine strain authentication, when available, because phenotypic examination is quick and inexpensive. Even if these markers are not practical as a unique strain identifier, in combination with other methods, they may limit the total number of steps necessary. We employed only phenotypic traits that had been observed over years in our laboratories and had been previously reported and described by other researchers. Development of new phenotypic markers as identifying characteristics requires extensive knowledge about the inheritance of characters. The traits employed in the MR4 authentication matrix follow.

Screening for genetic markers is more time consuming and costly than the previously mentioned methods. However, for many of the strains housed at the MR4, the value of the strain depends on fixed genetic characters; so this method of screening is logical and necessary. The first criteria for molecular characteristics in our scenario were those that were of distinct importance to the value of our strains such as the refractory nature of the thioester-containing protein gene for L3-5 or the 2La chromosomal arrangement for SUA2La. It was determined that these two established PCR methods (TEP1 [15] and 2La [16]) could be multiplexed reducing the time, cost, and effort (Figure 3). Similarly, SNPs commonly associated with the Mopti/Savanna (M/S) rDNA types were previously used to create a molecular assay combined with an established method for An. gambiae species identification assay as described in Wilkins et al. [17] (Figure 4). The white gene was also useful in separating genetically similar MOPTI, MALI and ZAN/U, PIMERPENA using strain-specific SNPs (Figure 5).

The colonies illustrated in this example have been maintained for many years and the traits tested extensively. For initial screenings of new colonies, it is recommended that 100% of the individuals be examined for each characteristic for no less than 5 sequential generations and continuing in a recurring pattern (every nth generation, based on laboratory constraints) to ensure stability and reproducibility of characters and testing mechanics. For each laboratory and situation, the quantity of individuals to test routinely should also be based on frequency of testing and laboratory constraints. For morphological traits or insecticide resistant strains, we screen 100% of the individuals every fifth generation due to ease of inspection. Screening all individuals provides the most confidence but is not necessarily imperative if testing occurs frequently. For molecular traits, no less than 48 individuals are tested every fifth generation in our laboratories. Required numbers were chosen somewhat arbitrarily by our group based on laboratory constraints, feasibility, and the animal of interest. However, recurring and frequent testing over the course of generations is most important.

An authentication record of laboratory strains is useful for historical assurance that uniqueness has not been compromised. Conversion of the chosen authentication methodologies into records is important. At the MR4, each line of Table 1 was converted into a stock-specific authentication form including dates, generation number, and specific criteria (how many individuals should be viewed, expected result, etc.). The observer is responsible for signing and dating a record log after the completion of each assay. If any result is incomplete or ambiguous, the process begins anew with the next generation. Once all assays are completed, the paperwork is archived and available for future researchers. A full evaluation of each strain should be conducted in a recurring fashion determined by the individual researcher. Storing individual insect samples after each authentication evaluation may be useful in the future for determining the timeline of any genetic changes. Archived material may also be valuable for future determinations of whether newly discovered alleles have always been present or if, and possibly when, a strain's genetic makeup may have changed, for example.

By employing an assortment of markers, an authentication matrix such as this one (Table 1, Figure 1) can be established to discriminate large or small numbers of strains. At the MR4, similar matrices are kept for all morphologically indistinguishable strains.

We analyzed Luminex technology as an alternative method for authentication. With this technology, large numbers of SNP sites can be simultaneously detected in a gene(s) [25]. By multiplexing a PCR with several genes of interest, we were able to create a successful Luminex procedure for screening samples for authentication (data not presented). However, the PCR plus Luminex procedure was more time consuming and expensive than the 1 or 2 PCR methods required as presented here. If more cost- or time-efficient, Luminex technology could be a valuable authentication tool, assuming adequate SNP sites were available for discrimination. The different genes of interest would need to be limited as well to avoid many preliminary PCR steps.

Allozyme analyses and karyotyping are successfully used to discriminate between conspecific laboratory strains [6,26,27]. These methods, however, require extensive training in sample preparation and analyses making routine usage difficult, especially if simpler, less expensive methods are available.

We also considered insertion patterns of transposable elements (TEs) (i.e. mobile genetic units capable of replicating and spreading in the host genome) which have recently been applied to study genetic differentiation between An. gambiae molecular forms [28-32]. Among TEs, Short INterspersed Elements (SINEs) have been extensively used as phylogenetic and population genetic markers in primate taxa and An. gambiae [28,30,32]. Despite the discriminative value for our strains detected in a few loci during preliminary research (data not presented), we did not pursue this approach for a number or reasons. Most loci tested were polymorphic in most strains and therefore only a few presented fixed variants with discriminative value. Moreover, most loci presented a high percentage of PCR failure (6-49%). For routine screenings, this was especially important as the absence of any amplification product for a particular sample could indicate the presence of a particular mutation in the primer binding sequence or simply a PCR failure. Also, using this method, common "stutter" bands and shadow bands made routine scoring quite difficult. In addition, the few strains that could be classified with this approach could also be authenticated using other techniques with greater reliability.

Mosquitoes were obtained from the MR4 (CDC Atlanta, GA USA, Table 2). Larvae were reared at 27°C using a standard method [33] with larval diet from Aquaricare™ (Koi Floating Blend). Two cohorts of approximately 300 second instar larvae from each strain were reared concurrently in white and black bottomed pans in order to elicit the homochromy response as described by Benedict et al [13].

Cohorts of 600 fourth instar larvae were treated with insecticides as described in the MR4 Methods in Anopheles Research Training Manual (section 4.3.1) [34] with final concentrations and exposure times as follows: Permethrin (1 ppm, 24 h), Propoxur (20 ppm, 1 h), Dieldrin (1 ppm, 1 h), DDT (0.4 ppm, 24 h).

Mosquito samples were prepared for PCR by the method of Rafferty et al [35]. PCR products were observed by separation on 0.5× TBE agarose gels run in 0.5× TBE buffer at 12 v/cm and fragment sizes were estimated using a 1 kb ladder marker (Invitrogen ^®). Thermal cycling for all analyses was performed in a Bio-Rad iCycler^®. Go-Taq DNA polymerase and the manufacturer's (Promega^®) recommended buffer at 1× concentration was used for all reactions. PCR reactions consisted of 0.75 U of Go-Taq polymerase, 1 mM MgCl₂, all primers at 1 μM, 0.08 mM dNTPs and a 25 μl total volume.

In order to obtain SNP sites to discriminate MOPTI from MALI and ZAN/U from PIMPERENA, the white gene from these mosquito strains was amplified using primers WG2 and WG5 and conditions as described by Mukabayire et al [37]. PCR products were sequenced and matched those of Genbank:U29485 with the exceptions of SNP sites shown in Table 4. SNP sites were used to design primers using the intentional mismatch primer (IMP) method [17] as shown in Table 3.