Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata

Kynurenine pathway is critically important to catabolize tryptophan, to produce eye chromes, and to protect nervous system in insects. However, several issues related to tryptophan degradation remain to be clarified. In the present paper, we identified three genes (karmoisin, vermilion and cardinal) involved in kynurenine pathway in Henosepilachna vigintioctopunctata. The karmoisin and cardinal were highly expressed in the pupae and adults having compound eyes. Consistently, high-performance liquid chromatography result showed that three ommochrome peaks were present in adult heads rather than bodies (thoraces, legs, wings and abdomens). RNA interference (RNAi)-aided knockdown of vermilion caused accumulation of tryptophan in both adult heads and bodies, disappearance of ommochromes in the heads and a complete loss of eye color in both pupae and adults. Depletion of cardinal brought about excess of 3-hydroxykynurenine and insufficient ommochromes in the heads and decolored eyes. RNAi of karmoisin resulted in a decrease in ommochromes in the heads, and a partial loss of eye color. Moreover, a portion of karmoisin-, vermilion- or cardinal-silenced adults exhibited negative phototaxis, whereas control beetles showed positive phototaxis. Furthermore, dysfunctions of tryptophan catabolism impaired climbing ability. Our findings clearly illustrated several issues related to kynurenine pathway and provided a new insight into the physiological importance of tryptophan catabolism in H. vigintioctopunctata.

     

Sub-cellular Localisation of the White/Scarlet ABC Transporter to Pigment Granule Membranes Within the Compound Eye of Drosophila Melanogaster

The white, scarlet, and browngenes of Drosophila melanogasterencode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of targeting eye color in Tenebrio molitor through RNA interference of tryptophan 2,3‐dioxygenase (vermilion): Implications for insect farming

The gene vermilion encodes tryptophan 2,3‐dioxygenase, part of the ommochrome pathway, and is responsible for the dark pigmented eyes in some insects, including beetles. Using RNA interference, we targeted the vermilion gene ortholog in embryos and pupae of the yellow mealworm, Tenebrio molitor, resulting in larvae and adults, respectively, that lacked eye pigment. RNA‐Seq was used to analyze the impact of vermilion‐specific RNA interference on gene expression. There was a 425‐fold reduction in vermilion gene expression (p = 0.0003), as well as significant (p < 0.05) differential expression of 109 other putative genes, most of which were downregulated. Enrichment analysis of Gene Ontology terms found in the differentially expressed data set included genes known to be involved in the ommochrome pathway. However, enrichment analysis also revealed the influence of vermilion expression on genes involved in protein translocation to the endoplasmic reticulum, signal transduction, G‐protein‐coupled receptor signaling, cell‐cycle arrest, mannose biosynthesis, and vitamin transport. These data demonstrate that knockdown of vermilion in T. molitor results in complete loss of eye color (white‐eyed phenotype) and identify other interrelated genes in the vermilion metabolic pathway. Therefore, a dominant marker system based on eye color can be developed for the genetic manipulation of T. molitor to increase the value of mealworms as an alternative food source by decreasing negative traits, such as disease susceptibility, and increasing desired traits, such as protein content and vitamin production.     

The ABCs of Eye Color in Tribolium castaneum: Orthologs of the Drosophila white,scarlet, and brown Genes

In Drosophila melanogaster, each of the three paralogous ABC transporters, White, Scarlet and Brown, is required for normal pigmentation of the compound eye. We have cloned the three orthologous genes from the beetle Tribolium castaneum. Conceptual translations of Tribolium white (Tcw), scarlet (Tcst), and brown (Tcbw) are 51, 48, and 32% identical to their respective Drosophila counterparts. We have identified loss-of-eye-pigment strains that bear mutations in Tcw and Tcst: the Tcw gene in the ivory (i) strain carries a single-base transversion, which leads to an E → D amino-acid substitution in the highly conserved Walker B motif, while the Tcst gene in the pearl (p) strain has a deletion resulting in incorporation of a premature stop codon. In light of these findings, the mutant strains i and p are herein renamed white^ivory (wⁱ) and scarlet^pearl (st^p), respectively. In addition, RNA inhibition of Tcw and Tcst recapitulates the mutant phenotypes, confirming the roles of these genes in normal eye pigmentation, while RNA interference of Tcbw provides further evidence that it has no role in eye pigmentation in Tribolium. We also consider the evolutionary implications of our findings.     

Metabolomic profiling of permethrin-treated Drosophila melanogaster identifies a role for tryptophan catabolism in insecticide survival

Insecticides and associated synergists are rapidly losing efficacy in target insect pest populations making the discovery of alternatives a priority. To discover novel targets for permethrin synergists, metabolomics was performed on permethrin-treated Drosophila melanogaster. Changes were observed in several metabolic pathways including those for amino acids, glycogen, glycolysis, energy, nitrogen, NAD⁺, purine, pyrimidine, lipids and carnitine. Markers for acidosis, ammonia stress, oxidative stress and detoxification responses were also observed. Many of these changes had not been previously characterized after permethrin exposure. From the altered pathways, tryptophan catabolism was selected for further investigation. The knockdown of some tryptophan catabolism genes (vermilion, cinnabar and CG6950) in the whole fly and in specific tissues including fat body, midgut and Malpighian tubules using targeted RNAi resulted in altered survival phenotypes against acute topical permethrin exposure. The knockdown of vermilion, cinnabar and CG6950 in the whole fly also altered survival phenotypes against chronic oral permethrin, fenvalerate, DDT, chlorpyriphos and hydramethylnon exposure. Thus tryptophan catabolism has a previously uncharacterized role in defence against insecticides, and shows that metabolomics is a powerful tool for target identification in pesticide research.     

Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm,Bombyx mori

ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.     

Pupal RNA interference methods for analyzing adult development in stag beetles

Holometabolous insects dramatically change their morphology via molt, both from larva to pupa and from pupa to adult. In nonmodel insects, RNA interference (RNAi) is a strong tool for analyzing gene function during postembryonic development. In many cases, larval RNAi is effective for analyzing genes involved in morphogenesis via metamorphosis. However, RNAi of genes involved in development sometimes results in lethality before animals metamorphose to pupae and/or adults, making it impossible to analyze their function during the pupal period. In this study, we establish a pupal RNAi system in the stag beetle Dorcus rectus. We selected the genes white and scarlet for RNAi knockdown to investigate appropriate injection timing and position. Both genes are known to be involved in eye pigmentation. By using these candidate genes, we demonstrate the potential of pupal RNAi in this experimental system. This method will be useful for analyzing pupal-specific morphogenesis including fine-shaping of the enlarged male mandible in this species.     

Cloning and characterization of thewhite andtopaz eye color genes from the sheep blowflylucilia cuprina

Summary Clones carrying thewhite andtopaz eye color genes have been isolated from genomic DNA libraries of the blowflyLucilia cuprina using cloned DNA from the homologouswhite andscarlet genes. respectively, ofDrosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of thewhite gene ofL. cuprina has been determined, and the homology to the corresponding region ofD. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing theD. melanogaster genes is free of repeated sequences. that encompassing theirL. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome ofL. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 10⁴ copies to less than 10² copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both thewhite andtopaz gene regions are found within and between populations ofL. cuprina.     

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.     

Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster

Molecular and cellular evidence argues that a heterodimer between two ABC transporters, the White protein and the Brown protein, is responsible for pumping guanine into pigment‐synthesizing cells of the fruit fly, Drosophila melanogaster. Previous studies have not detected White or Brown outside pigment‐synthesizing cells nor have behavioral effects of null mutants been reported, other than those that are visually dependent. Nevertheless, we show here that exposure to the volatile general anesthetic (VGA) enflurane reveals a difference in neuromuscular performance between wild‐type flies and those that carry a null allele in either the white or brown gene. Specifically, in a test of climbing ability, w¹¹¹⁸ or bw¹ flies are much less affected by enflurane than are congenic controls. Altered anesthetic sensitivity is still observed when visual cues are reduced or eliminated, arguing that white and brown contribute to neural function outside the eye. This hypothesis is supported by the detection of white message in heads of flies that are genetically altered so as to lack pigment‐producing cells. The w¹¹¹⁸ or bw¹ mutations also alter the response to a second VGA, halothane, albeit somewhat differently. Under some conditions, the combination of w¹¹¹⁸ with another mutation that affects anesthesia leads to a drastically altered phenotype. We consider several ways by which diminished transport of guanine could influence neural function and anesthetic sensitivity. Published 2001 John Wiley & Sons, Inc. J Neurobiol 49: 339–349, 2001     

The effect of eye reduction on White position-effect variegation in Drosophila melanogaster

Position-effect variegation for the white locus was studied in normally shaped eyes and in reduced eyes of Bar (B) and Drop (Dr) flies. The average number of spots per eye is successively lower in +, B, and Dr eyes; moreover, B eyes show a relatively strong pigmentation. No simple relation seems to be present between the degree of pigmentation and the number of facets, either between +, B, and Dr eyes or within classes of Dr eyes that have been analysed.The chance that ommatidia will become pigmented follows a gradient across mottled eyes of wild-type shape that seems fixed early in development. The gradient is less clear or absent in B eyes.The results are best interpreted on the basis of the cell-lineage theory and an early one-sided action of B on the developing eye disc after fixation of the gradient.     

Effect of mutation-induced excess brain concentration of intermediates of the kynurenine pathway of tryptophan metabolism on stress resistance of courtship behavior and communicative sound production in male Drosophila melanogaster

The resistance of courtship behavior and communicative sound production to heat shock (37°C, 30 min) was studied in wild-type Canton S (CS) male Drosophila melanogaster and males of two strains with defects in the kynurenine pathway of tryptophan metabolism (KPTM) caused by mutations cinnabar (block at the level of kynurenine-3-hydroxylase leading to accumulation of kynurenic acid, a neuroprotective metabolite, in the brain) and cardinal (block at the level of phenoxazinone synthetase causing accumulation of 3-hydroxykynurenine, an oxidative stress generator, in the brain). Males of each strain were divided into four groups. Males from control groups were not exposed to heat shock. The other groups were exposed to heat shock at the late embryonic/early larval (the first instar) developmental stage, when mushroom bodies are formed (HS1 groups); at the prepupal stage, when the brain central complex develops (HS2 groups); or at the imago stage 1 h before the experiment (HS groups). All males were tested at an age of five days. Virgin and fertilized five-day-old CS females served as courtship objects. The courtship behavior and singing of control CS and cinnabar males were similar. Control cardinal males also had high motivation, but their courtship efficiency was lower because of less precise movements (wing vibration was often not accompanied by sound production) and hyperexcitability. Exposure of first-instar larvae to heat shock had almost no effect on behavior or singing of adult males of any strain. In cardinal males exposed to heat shock at the prepupal stage or, especially, at the imago stage 1 h before the test (the HS2 and HS groups), courtship was strongly impaired, and various distortions appeared in their sound signals, which indicated disturbance of coordination between elements of the song center and their interaction with pacemakers. These effects were much milder or absent altogether in HS2 and HS wild-type males and, especially, cinnabar males. Thus, permanent excess of 3-hydroxykynurenine in the male brain dramatically decreased their stress resistance. In contrast, excess of kynurenic acid alleviated the consequences of stress.     

Morphological and molecular development of the eyes during embryogenesis of the freshwater planarian Schmidtea polychroa

Photoreception is one of the most primitive sensory functions in metazoans. Despite the diversity of forms and components of metazoan eyes, many studies have demonstrated the existence of a common cellular and molecular basis for their development. Genes like pax6, sine oculis, eyes absent, dachshund, otx, Rx and atonal are known to be associated with the specification and development of the eyes. In planarians, sine oculis, eyes absent and otxA play an essential role during the formation of the eye after decapitation, whereas pax6, considered by many authors as a master control gene for eye formation, does not seem to be involved in adult eye regeneration. Whether this is a peculiarity of adult planarians or, on the contrary, is also found in embryogenesis remains unknown. Herein, we characterize embryonic eye development in the planarian species Schmidtea polychroa using histological sections and molecular markers. Additionally, we analyse the expression pattern of the pax6–sine oculis–eyes absent–dachshund network, and the genes Rx, otxA, otxB and atonal. We demonstrate that eye formation in planarian embryos shows great similarities to adult eye regeneration, both at the cellular and molecular level. We thus conclude that planarian eyes exhibit divergent molecular patterning mechanisms compared to the prototypic ancestral metazoan eye.