Can there be a common, risk-based framework for decisions around live insect trade?

A network of scientists involved in shipment of live insects has met and generated a series of articles on issues related to live insect transport. The network is diverse, covering large-scale commercial interests, government operated areawide control programmes, biomedical research and many smaller applications, in research, education and private uses. Many insect species have a record of safe transport, pose minimal risks and are shipped frequently between countries. The routine shipments of the most frequently used insect model organism for biomedical research, Drosophila melanogaster, is an example. Successful large scale shipments from commercial biocontrol and pollinator suppliers also demonstrate precedents for low-risk shipment categories, delivered in large volumes to high quality standards. Decision makers need access to more information (publications or official papers) that details actual risks from the insects themselves or their possible contaminants, and should propose proportionate levels of management. There may be harm to source environments when insects are collected directly from the wild, and there may be harm to receiving environments. Several risk frameworks include insects and various international coordinating bodies, with experience of guidance on relevant risks, exist. All stakeholders would benefit from an integrated overview of guidance for insect shipping, with reference to types of risk and categories of magnitude, without trying for a single approach requiring universal agreement. Proposals for managing uncertainty and lack of data for smaller or infrequent shipments, for example, must not disrupt trade in large volumes of live insects, which are already supporting strategic objectives in several sectors.

À l'occasion d'une réunion d'experts, un réseau de chercheurs travaillant sur l'expédition d'insectes vivants a produit un ensemble d'articles traitant des questions liées au transport des insectes vivants. Ce réseau est diversifié et représente un large éventail d'intérêts privés et de programmes de lutte biologique à grande échelle menés par les pouvoirs publics, en plus du secteur de la recherche biomédicale et de nombreux acteurs intervenant dans des applications de plus petite envergure relevant de la recherche, de l'enseignement ou du secteur privé. Un grand nombre d'espèces d'insectes sont transportées en toute sécurité avec un niveau de risque minimal, y compris lors des fréquentes expéditions internationales. Un exemple de ces expéditions régulières concerne l'espèce la plus utilisée par la recherche biomédicale en tant qu'organisme modèle, à savoir Drosophila melanogaster. Les exemples réussis d'expéditions à grande échelle provenant de fournisseurs d'agents de lutte biologique et de pollinisateurs produits par le secteur privé offrent également un relevé documenté des diverses catégories d'expéditions à faible risque pour des livraisons d'insectes vivants en grandes quantités et répondant à des normes de qualité élevées. Les décideurs politiques devraient pouvoir accéder à plus d'informations (à travers des publications ou des articles officiels) décrivant en détail les risques réels associés aux insectes eux-mêmes ou à leurs contaminants éventuels, et proposer en connaissance de cause des niveaux de gestion proportionnels à ces risques. La récolte d'insectes prélevés directement de la nature peut être dommageable aussi bien pour les environnements source que pour ceux de destination. Plusieurs cadres fondés sur le risque intègrent désormais les insectes dans leurs directives. Par ailleurs, nombre d'organismes internationaux de coordination ont acquis une expérience dans l'élaboration de lignes directrices face à ces risques. Il serait bénéfique pour toutes les parties prenantes de disposer d'une vue d'ensemble intégrée des directives applicables aux expéditions d'insectes, qui recense les différents types de risque et leurs ordres de grandeur sans se prononcer sur une approche unique qui nécessiterait une adhésion universelle. Ni les propositions visant à gérer l'incertitude ni l'insuffisance des données disponibles sur les expéditions d'insectes en petits nombres ou occasionnelles ne doivent perturber les échanges commerciaux d'insectes vivants en grandes quantités, échanges qui participent aujourd'hui aux objectifs stratégiques de nombreux secteurs.

Una red de científicos relacionados de un modo u otro con el transporte de insectos vivos mantuvo un encuentro y generó una serie de artículos en torno a la cuestión. En la red, muy heterogénea, convergen desde intereses comerciales de gran calado hasta programas públicos de lucha biológica en grandes territorios, pasando por la investigación biomédica y por numerosas aplicaciones de menor dimensión en ámbitos como la investigación, la enseñanza u otros usos privados. Muchas especies de insectos cuentan con un buen historial de seguridad en el transporte, presentan un riesgo mínimo y son expedidas a menudo de un país a otro. Buen ejemplo de ello son los envíos sistemáticos de ejemplares de Drosophila melanogaster, que es el insecto utilizado con más frecuencia como organismo modelo en la investigación biomédica. Otro precedente de envíos voluminosos cuyo transporte y entrega se ajusta a los más exigentes criterios de calidad lo sientan las remesas de grandes cantidades de polinizadores y agentes de control biológico remitidos por proveedores comerciales. Los responsables de adoptar decisiones deben disponer de más y más detallada información (publicaciones o documentos oficiales) sobre los riesgos reales derivados de los propios insectos o sus posibles contaminantes para proponer a partir de ahí medidas de gestión proporcionadas. Los perjuicios ambientales pueden darse tanto en origen (cuando hay captura salvaje de insectos, o sea recolección directa en el medio) como en destino. Hay varios sistemas de determinación del riesgo que incluyen a los insectos y también existen diversos organismos de coordinación internacional que ya tienen experiencia en sentar pautas sobre los riesgos en la materia. Todos los interlocutores del sector se beneficiarían de una visión global e integrada del transporte de insectos, que remita a diferentes tipos de riesgo y categorías de magnitud, sin necesidad de buscar un sistema único que exija consenso universal. Las propuestas para gestionar la incertidumbre y la falta de información en el caso de envíos infrecuentes o de pequeñas dimensiones, por ejemplo, no deben interferir en el transporte de grandes cantidades de insectos vivos, que ya está sirviendo a objetivos estratégicos en varios sectores.

An inexpensive and effective larval diet for Anopheles arabiensis (Diptera: Culicidae): eat like a horse, a bird, or a fish?

A successful sterile insect technique program depends upon mass production of good-quality sterile insects for release into a target area. Specifically, to control Anopheles arabiensis Patton (Diptera: Culicidae) in a pilot area in northern Sudan, 1 million An. arabiensis sterile males per day are needed. To reach this production objective, mosquito mass rearing is indispensable and larval diet, a key parameter for the production of healthy male mosquitoes, needs to be cost-effective. The Koi Floating Blend fish food, previously used at the Food and Agriculture Organization/International Atomic Energy Agency Insect Pest Control Laboratory for the routine rearing of the An. arabiensis colony, is no longer available. The aim of this study was to find a cheap and effective substitute for the discontinued diet. Several candidate powdered diets that are commercially available were tested to determine the best diet. By using mixture experiment principles and response surface methodology, the combination of two components, bovine liver powder and tuna meal, showed the best results in terms of larval survival, developmental, rate and adult size. The addition of a vitamin mixture further improved the diet. These positive production results coupled with the relatively low cost of our blend demonstrated the possibility of its use for mass rearing purpose.

Comparisons of life-history characteristics of a genetic sexing strain with laboratory strains of Anopheles arabiensis (Diptera: Culicidae) from northern Sudan.

A genetic sex separation strain (GSS) has been created for Anopheles arabiensis (Patton) (Diptera: Culicidae), one of the major African malaria vectors, for use in controlling wild populations of this species via the sterile insect technique (SIT). This GSS strain, "ANO IPCL1," allows sex separation by a translocation linking a dieldrin resistance allele and the Y chromosome. Differences between ANO IPCL1 relative to wild strains might reflect its field performance and therefore are of concern. Of more immediate interest is how differences might affect production during mass rearing. Life-history parameters were measured for the ANO IPCL1 strain and the two wild strains from which it originated. Although developmental rate differences were found among them, none were large. However, a major observed variation was the very low intrinsic fertility of ANO IPCL1 because of the translocation itself. This resulted in a much lower rate of increase: ANO IPCL1 was able to double its population size, in 7.8 +/- 0.4 d, whereas Dongola and Sennar strains could do so in 4.9 +/- 0.5 and 5.6 +/- 0.4 d. The presence of the Y-autosome translocation mainly affected the natural fertility of the males, and this will require amplification steps during mass rearing.

A new larval tray and rack system for improved mosquito mass rearing.

The requirement for efficient mosquito mass rearing technology has been one of the major obstacles preventing the large scale application of the Sterile Insect Technique against mosquitoes. At the Food and Agriculture Organization/International Atomic Energy Agency (FAO/ IAEA) Insect Pest Control Laboratories we developed a larval rearing unit based on the use of a stainless steel rack that operates 50 thermoformed ABS plastic trays and is expected to be able to successfully rear 140,000-175,000 Anopheles arabiensis (Patton) adult mosquitoes per rack. The mechanized rearing unit is simple to handle, maintains minimal water temperature variation and negligible water evaporation and allows normal larval development. The mosquito mass-rearing tray was designed to provide a large surface area of shallow water that would closely mimic natural breeding sites. The trays stack into a dedicated rack structure and filling and draining were easily performed. The close stacking of the trays in the rack and the possibility to tightly line up several racks makes this rearing unit a valid solution for maximal use of the space thus reducing construction, heating, and cooling costs. The low amount of labor required to operate the system also reduces labor costs that represent one of the main expenditures in any mass rearing facility operation. Preliminary experiments performed on Aedes albopictus (Skuse) also confirm the possibility of successfully extending the use of this technology to other mosquito species. Our larval rearing unit could enhance any mosquito control strategy in which large-scale releases of mosquitoes are needed to suppress or replace natural populations.

Infertility resulting from transgenic I-PpoI male Anopheles gambiae in large cage trials.

OBJECTIVES: Anopheles gambiae is the primary vector of malaria in sub-Saharan Africa and is a potential target of genetic control programs. We determined the capacity of male A. gambiae created by germline transformation to introduce infertility into stable age-distribution populations. We also determined effects of the transgenes on life history. METHODS: Stable age-distribution populations of A. gambiae mosquitoes were established in large indoor cages. Male mosquitoes carrying an I-PpoI homing endonuclease gene were introduced at ×5 and ×10 release rates where they competed with target male mosquitoes for matings. Similar trials were conducted in small cages with an additional ×1 release level. RESULTS: Infertility was successfully introduced into all target populations. In supporting experiments, complete female infertility was observed in all strains and species of the A. gambiae complex to which transgenic males were mated. Life history experiments demonstrated that reductions in I-PpoI male vigor exist in the form of reduced adult male emergence, longevity and competitiveness. DISCUSSION: A. gambiae I-PpoI males are capable of introducing high levels of infertility in target populations in indoor cage trials. This was accomplished despite losses of vigor resulting from the HEG transgene. These results motivate further trials of sexually I-PpoI A. gambiae in outdoor cage and field trials.

Density-dependent effects in experimental larval populations of Anopheles arabiensis (Diptera: Culicidae) can be negative, neutral, or overcompensatory depending on density and diet levels.

Anopheles arabiensis Patton (Diptera: Culicidae) larvae were reared from hatching to the adult stage in the laboratory under a range of diet and larval concentrations using a factorial design. The range circumscribed most of the larval densities and diet concentrations that would allow larval growth and survival using the particular diet formulation and water volume we tested. We determined how these variables affected three outcomes, as follows: larval development rate, survival, and wing length. As has been reported previously, negative density dependence of survival as a function of increased larval density was the prevalent effect on all outcomes when diet was limiting. When diet was not limiting, density dependence was not observed, and three cases of overcompensatory survival were seen. We discuss these results in the context of diet and larval densities for mass rearing and the effect of larval competition on control strategies.

Relationship of larval desiccation to Anopheles gambiae Giles and An. arabiensis Patton survival.

The relationship between mosquito 4th instar larval desiccation and survival to adulthood was explored by three methods in the laboratory. Two colonies of Anopheles arabiensis and one of Anopheles gambiae were studied. We found significant differences in tolerance to desiccation among all three stocks suggesting an intra- and interspecific genetic component to desiccation tolerance. An. arabiensis KGB, originating from Zimbabwe about 1975, had a much-reduced desiccation tolerance compared to An. gambiae G3, colonized in the Gambia in 1975, and An. arabiensis DONGOLA which originated in Sudan in 2004. Individuals of the G3 stock survived desiccation of times up to 40 min with survival of 0.52. The degree of difference in tolerance between G3 and DONGOLA was smallest and was detected by one of three experimental methods. Mass losses of individuals that were weighed individually and survived to adulthood averaged 27% and 29% for G3 and DONGOLA and 20% for the less tolerant KGB stock, respectively. Such differences in survival in transiently dry larval habitats may account in part for differences in the distribution of these species and karyotypes.

X and Y chromosome inheritance and mixtures of rDNA intergenic spacer regions in Anopheles gambiae.

We observed Anopheles gambiae sensu stricto stocks that contained both Mopti (M) and Savanna (S) rDNA intergenic spacers (IGS). ASEMBO1 male IGS sequences consistently had a mixture. A diagnostic M and S Hha I restriction enzyme site in these fragments was concordant with two SNPs associated with M and S. Standard M and S stocks demonstrated X-chromosome-only inheritance of the rDNA form, but the ASEMBO1 males showed X and Y chromosome linkage of mixed rDNA. The metaphase Y chromosomes of ASEMBO1 contained a significantly larger amount of DNA relative to the X than a standard S stock. Analysis of wild A. gambiae males from the putative location of origin of the ASEMBO1 stock did not detect the same pattern of polymorphism observed in the laboratory stock but did detect heterogeneous arrays including some missing a diagnostic Hha I restriction site. These results demonstrate that M and S IGS types can occur within the rDNA arrays of a chromatid in laboratory A. gambiae stocks, and some A. gambiae s.s. have rDNA on the Y chromosome.

Sex separation of tsetse fly pupae using near-infrared spectroscopy.

Implementation of the sterile insect technique for tsetse (Glossina spp.) requires that only sterile male insects be released; thus, at some stage of the fly production process the females have to be removed. A further constraint in the use of the sterile insect technique for tsetse is that the females are needed for colony production and hence, a non-destructive method of sex separation is required. In most tsetse sterile insect technique programmes thus far, females have been eliminated from the released material by hand-separation of chilled adults. Using near-infrared (NIR) spectroscopy, significant differences have been found between the spectra for the pupae of male and female G. pallidipes Austen. Significantly, the differences appear to be maximized 4-5 days before emergence of the adults. Tsetse fly pupae up to five days before emergence can be sexed with accuracies that generally range from 80 to 100%. This system, when refined, will enable effective separation of male and female pupae to be carried out, with emerged females being returned to the colony and males being irradiated and released. If separation can be achieved five days before emergence, this will also enable irradiated male pupae to be shipped to other destinations as required. Other Diptera were evaluated using this system but had lower classification accuracies of 50-74%. This may be due to the difference in reproductive physiology between these different fly groups.

Independent mutations in the Rdl locus confer dieldrin resistance to Anopheles gambiae and An. arabiensis.

Substitutions of a conserved alanine residue in the Rdl locus coding for a gamma-aminobutyric acid (GABA) receptor subunit with serine or glycine confer resistance to dieldrin in various insect species. Here, we show that alanine to glycine substitution in the Rdl locus of the malaria vector, Anopheles gambiae, is genetically linked to resistance to dieldrin. An alanine to serine substitution developed independently in a dieldrin resistant strain of An. arabiensis. An allele-specific polymerase chain reaction (PCR) assay was able to differentiate dieldrin resistant and susceptible mosquitoes.

Genetic traits of the mosquito Anopheles gambiae: red stripe, frizzled, and homochromy1.

The expression, inheritance, and linkage relationships of three genetic traits were studied in the malaria vector Anopheles gambiae. Red stripe (Rs) is a common phenotypic polymorphism in numerous A. gambiae populations, whereas frizzled (f) and homochromy1 (hom1) were isolated from (60)Co-irradiated mosquitoes. Red stripe appears as a diffuse stripe of pigment on the dorsum of larvae and pupae and is variable in expressivity and penetrance. Our data demonstrate that Red stripe results from a heterozygous collarless genotype (i.e., c+ c, chromosome 2) and is essentially sex-limited to females. frizzled is a sex-linked recessive semi-lethal identified by deformed lateral larval setae; its lethality manifests as low rates of adult emergence and brief adult survival. frizzled is located on the X chromosome between pink eye and Mosaic, 3 cM from Mosaic and approximately 12 cM from pink eye. Finally, the mutation homochromy1 (hom1) is on chromosome 2 and causes a recessive phenotype that prevents normal darkening of larvae when reared in a black container. Unlike mutants with this characteristic described thus far, the eye color of hom1 mutants is normal. We determined that hom1 is located between Dieldrin resistance and collarless, approximately 3 cM from the latter. We discuss the possibility of differences in male and female recombination values and the range of values that have been observed in test crosses for chromosome 2 markers.

Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element.

Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) injected into mosquito embryos. Two G1 generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event in the injected G0 embryos. Genetic cross data suggest that the insertion site of the element either resulted in, or is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios in outcrosses to non-transformed individuals and failure to establish a pure-breeding line. The insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement in the injection technique at the end of the studies resulted in increased G0 hatching, transient expression and EGFP-expression rates among G1 progeny.

Mosquito collections following local transmission of Plasmodium falciparum malaria in Westmoreland County, Virginia.

A 63-year-old woman from Colonial Beach, Westmoreland County, VA, was diagnosed with Plasmodium falciparum malaria on July 19, 1998. The woman had no history of international travel, intravenous drug use, blood transfusion, or other risk factor for contracting the disease. She seldom left the county and generally spent her evenings indoors, leading to the conclusion that she had been bitten locally by an infected mosquito. Colonial Beach is host to a population of migrant agricultural laborers from areas in which malaria occurs, but a blood survey of 89 Haitians and Mexicans failed to find Plasmodium parasites, specific antibodies, or clinical cases of malaria. Mosquito surveys were conducted during 2 days (July 22 and 28, 1998) with carbon-dioxide-baited light traps, larval and pupal collections, and landing collections. Thirteen species of mosquitoes were identified morphologically, including 4 potential vectors: Anopheles crucians, An. punctipennis, An. smaragdinus (new state record), and An. quadrimaculatus s.s. (new state record). Identifications of the latter 2 species were confirmed by sequencing of the ITS2 DNA region from adults reared from locally collected larvae. Anopheles smaragdinus was the most common biting species among the potential vectors, although An. crucians was the most abundant in other kinds of collections. In addition, Ae. albopictus was collected in Westmoreland County for the 1st time.

The piggyBac element is capable of precise excision and transposition in cells and embryos of the mosquito, Anopheles gambiae.

The piggyBac transposable element was tested for transposition activity in plasmid-based excision and inter-plasmid transposition assays to determine if this element would function in Anopheles gambiae cells and embryos. In the Mos55 cell line, precise excision of the piggyBac element was observed only in the presence of a helper plasmid. Excision occurred at a rate of 1 event per 1000 donor plasmids screened. Precise excision of the piggyBac element was also observed in injected An. gambiae embryos, but at a lower rate of 1 excision per 5000 donor plasmids. Transposition of the marked piggyBac element into a target plasmid occurred in An. gambiae cells at a rate of 1 transposition event per 24,000 donor plasmids. The piggyBac element transposed in a precise manner, with the TTAA target site being duplicated upon insertion, in 56% of transpositions observed, and only in the presence of the piggyBac helper. The remaining transpositions resulted in a deletion of target sequence, a novel observation for the phenomenon of piggyBac element insertion. 'Hot spots' for insertion into the target plasmid were observed, with 25 of 34 events involving one particular site. These results are the first demonstration of the precise mobility of piggyBac in this malaria vector and suggest that the lepidopteran piggyBac transposon is a candidate element for germline transformation of anopheline mosquitoes.

Mosaic: a position-effect variegation eye-color mutant in the mosquito Anopheles gambiae.

The Mosaic (Mos) mutation, isolated in the F1 of 60Co-irradiated mosquitoes, confers variegated eye color to third and fourth instar larvae, pupae, and adults of the mosquito Anopheles gambiae. Mos is recessive in wild pink eye (p+) individuals, but is dominant and confers areas of wild-type pigment in mutant pink eye backgrounds. Mos is located 14.4 cM from pink eye on the X chromosome and is associated with a duplication of division 2B euchromatin that has been inserted into division 6 heterochromatin. Various combinations of Mos, pink eye alleles, and the autosomal mutation red eye were produced. In all cases, the darker pigmented regions of the eye in Mos individuals show the phenotypic interactions expected if the phenotype of those regions is due to expression of a p+ allele. Expression of Mos is suppressed by rearing larvae at 32 degrees C relative to 22 degrees C. All of these characteristics are consistent with Mos being a duplicated wild copy of the pink eye gene undergoing position-effect variegation.

A new marker, black, a useful recombination suppressor, In(2)2, and a balanced lethal for chromosome 2 of the mosquito Anopheles gambiae.

A new marker for the second chromosome of Anopheles gambiae, black, was isolated from progeny of 60Co-irradiated mosquitoes. The black mutation increases melanization of larval setae and portions of the cuticle that are heavily sclerotized such as the saddle and head capsule. Adults have a sooty color that almost completely eliminates white banding on wings, tarsi, and palps. Fertility and general vigor of black individuals is reduced relative to wild-type; however, this does not prevent routine use for genetic crossing. The black marker was mapped to an interval on chromosome 2 between collarless and Dieldrin resistance 22 centiMorgans (cM) from collarless and 39 cM from Dieldrin resistance. We also isolated from 60Co-irradiated mosquitoes a pericentric inversion, In(2)2, that was marked with dominant alleles of the independently assorting genes collarless and Dieldrin resistance. This inversion is in coupling with the pericentric inversion 2Rd and covers approximately two-thirds of chromosome 2 from divisions 9 to 22. While inbreeding In(2)2 heterozygotes, we isolated a stock in which the inversion was in repulsion to a chromosome marked with c b DlS and an unidentified recessive lethal. This arrangement produced a useful and stable chromosome 2 balancer system that has remained intact for 26 generations without selection. These genetic tools will reduce the effort requires to isolate, among other things, the genetic factors affecting malaria parasite interactions with the mosquito host.

Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly.

The mosquito Aedes aegypti is the world's most important vector of yellow fever and dengue viruses. Work is currently in progress to control the transmission of these viruses by genetically altering the capacity of wild Ae. aegypti populations to support virus replication. The germ-line transformation system reported here constitutes a major advance toward the implementation of this control strategy. A modified Hermes transposon carrying a 4.7-kb fragment of genomic DNA that includes a wild-type allele of the Drosophila melanogaster cinnabar (cn) gene was used to transform a white-eyed recipient strain of Ae. aegypti. Microinjection of preblastoderm mosquito embryos with this construct resulted in 50% of the emergent G0 adults showing some color in their eyes. Three transformed families were recovered, each resulting from an independent insertion event of the cn+-carrying transposon. The cn+ gene functioned as a semidominant transgene and segregated in Mendelian ratios. Hermes shows great promise as a vector for efficient, heritable, and stable transformation of this important mosquito vector species.

The Anopheles gambiae tryptophan oxygenase gene expressed from a baculovirus promoter complements Drosophila melanogaster vermilion.

An Anopheles gambiae cDNA encoding tryptophan oxygenase was placed under the control of the constitutive baculovirus promoter, ie-1. The chimeric construct, expressed transiently in vermilion (tryptophan oxygenase) mutants of Drosophila melanogaster, partially rescued adult eye color. The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product and the effectiveness of the ie-1 promoter in directing expression of foreign genes in live insects. The functionality of An. gambiae tryptophan oxygenase in a higher fly fulfils predictions based on its structural conservation throughout millions of years of independent evolution.

The Anopheles albimanus white gene: molecular characterization of the gene and a spontaneous white gene mutation.

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus.

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti.

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.