Functional characterization of olfactory receptors in three Dacini fruit flies (Diptera: Tephritidae) that respond to 1-nonanol analogs as components in the rectal glands

Dacini fruit flies (Tephritidae: Diptera), including destructive pest species, are strongly affected in their reproductive behaviors by semiochemicals. Notably, male lures have been developed for pest management e.g., aromatic compounds for the Oriental fruit fly Bactrocera dorsalis and the melon fruit fly Zeugodacus cucurbitae; terpenic α-ionone analogs for the solanaceous fruit fly, B. latifrons. Other than those specific male attractants, 1-nonanol analogs have been noticed as major aliphatic components in the male rectal gland, which is considered as a secretory organ of male sex pheromones. Although multiple semiochemicals associated with the life cycle of Dacini fruit flies have been identified, their behavioral role(s) and chemosensory mechanisms by which the perception occurs have not been fully elucidated. In this study, we conducted RNA sequencing analysis of the chemosensory organs of B. latifrons and Z. cucurbitae to identify the genes coding for chemosensory receptors. Because the skeletons of male attractants are different among Dacini fruit fly species, we analyzed phylogenetic relationships of candidate olfactory receptors (ORs) among the three species. We found that the OR phylogeny reflects the taxonomic relationships of the three species. We further characterized functional properties of OR74a in the three Dacini species to the 1-nonanol analogs related to components in the rectal glands. The three OR74a homologs responded to 1-nonanol, but their sensitivities differed from each other. The OR74a homologs identified from B. dorsalis and Z. cucurbitae responded significantly to 6-oxo-1-nonanol, but not to 1, 3-nonanediol and nonyl acetate, indicating similar binding properties of the homologous ORs

Characterization of candidate intermediates in the Black Box of the ecdysone biosynthetic pathway in Drosophila melanogaster: Evaluation of molting activities on ecdysteroid-defective larvae

Early steps of the biosynthetic pathway of the insect steroid hormone ecdysone remains the “Black Box” wherein the characteristic ecdysteroid skeleton is built. 7-Dehydrocholesterol (7dC) is the precursor of uncharacterized intermediates in the Black Box. The oxidation step at C-3 has been hypothesized during conversion from 7dC to 3-oxo-2, 22, 25-trideoxyecdysone, yet 3-dehydroecdysone is undetectable in some insect species. Therefore, we first confirmed that the oxidation at C-3 occurs in the fruitfly, Drosophila melanogaster using deuterium-labeled cholesterol. We next investigated the molting activities of candidate intermediates, including oxidative products of 7dC, by feeding-rescue experiments for Drosophila larvae in which an expression level of a biosynthetic enzyme was knocked down by the RNAi technique. We found that the administration of cholesta-4, 7-dien-3-one (3-oxo-Δ4, 7C) could overcome the molting arrest of ecdysteroid-defective larvae in which the expression level of neverland was reduced. However, feeding 3-oxo-Δ4, 7C to larvae in which the expression levels of shroud and Cyp6t3 were reduced inhibited molting at the first instar stage, suggesting that this steroid could be converted into an ecdysteroid-antagonist in loss of function studies of these biosynthetic enzymes. Administration of the highly conjugated cholesta-4, 6, 8(14)-trien-3-one, oxidized from 3-oxo-Δ4, 7C, did not trigger molting of ecdysteroid-defective larvae. These results suggest that an oxidative product derived from 7dC is converted into ecdysteroids without the formation of this stable conjugated compound. We further found that the 14α-hydroxyl moiety of Δ4-steroids is required to overcome the molting arrest of larvae in loss of function studies of Neverland, Shroud, CYP6T3 or Spookier, suggesting that oxidation at C-14 is indispensable for conversion of these Δ4-steroids into ecdysteroids via 5β-reduction

Predominant accumulation of a 3-hydroxy-γ-decalactone in the male rectal gland complex of the Japanese orange fly, Bactrocera tsuneonis

The Japanese orange fly, Bactrocera tsuneonis, infests various citrus crops. While male pheromone components accumulated in the rectal glands are well characterized for Bactrocera, but information regarding the chemical factors involved in the life cycles of B. tsuneonis remains scarce. Herein, several volatile chemicals including a γ-decalactone, (3R, 4R)-3-hydroxy-4-decanolide [(3R, 4R)-HD], were identified as major components, along with acetamide and spiroketals as minor components in the rectal gland complexes of male B. tsuneonis flies. The lactone (3R, 4R)-HD was also identified in female rectal gland complexes. The amount of this compound in mature males was significantly higher than those observed in females and immature males. The lactone (3R, 4R)-HD was detected in flies fed with sucrose only, indicating that this lactone is not derived from dietary sources during adulthood, but biosynthesized in vivo. The predominant accumulation of (3R, 4R)-HD in mature males also suggests a possible role in reproductive behavior

Historical perspective on the synonymization of the four major pest species belonging to the Bactrocera dorsalis species complex (Diptera, tephritidae)

An FAO/IAEA-sponsored coordinated research project on integrative taxonomy, involving close to 50 researchers from at least 20 countries, culminated in a significant breakthrough in the recognition that four major pest species, Bactrocera dorsalis, B. philippinensis, B. papayae and B. invadens, belong to the same biological species, B. dorsalis. The successful conclusion of this initiative is expected to significantly facilitate global agricultural trade, primarily through the lifting of quarantine restrictions that have long affected many countries, especially those in regions such as Asia and Africa that have large potential for fresh fruit and vegetable commodity exports. This work stems from two taxonomic studies: a revision in 1994 that significantly increased the number of described species in the Bactrocera dorsalis species complex; and the description in 2005 of B. invadens, then newly incursive in Africa. While taxonomically valid species, many biologists considered that these were different names for one biological species. Many disagreements confounded attempts to develop a solution for resolving this taxonomic issue, before the FAO/IAEA project commenced. Crucial to understanding the success of that initiative is an accounting of the historical events and perspectives leading up to the international, multidisciplinary collaborative efforts that successfully achieved the final synonymization. This review highlights the 21 year journey taken to achieve this outcome

Chemical ecology of insect-plant interactions: ecological significance of plant secondary metabolites.

Plants produce a diverse array of secondary metabolites as chemical barriers against herbivores. Many phytophagous insects are highly adapted to these allelochemicals and use such unique substances as the specific host-finding cues, defensive substances of their own, and even as sex pheromones or their precursors by selectively sensing, incorporating, and/or processing these phytochemicals. Insects also serve as pollinators often effectively guided by specific floral fragrances. This review demonstrates the ecological significance of such plant secondary metabolites in the highly diverse interactions between insects and plants

Exploitation of new attractants for fruit fly pests of economic importance

The fruit fly tribe Dacini (Tephritidae: Diptera) comprises more than 700 species including many fruit pests of economic importance, particularly in the two genera, Bactrocera and Zeugodacus. Males of many dacine species show strong affinity either to methyl eugenol (ME) or cue-lure (CL). However, there are over a hundred species that show no affinity to these lures. We have been seeking new attractants for these ‘nonresponding’ species particularly of endemic agricultural importance based on the following three criteria, (1) by observing their mating sequences, (2) their natural interactions with plants and (3) by analyzing specific chemicals the wild male flies accumulate in their secretory organ (rectal gland) associated with pheromone production. Orchid flowers of many Bulbophyllum species attract fruit fly males by emitting specific volatiles for pollination, in the rain forests of Southeast Asia. Several species produce either ME or raspberry ketone (RK), and some species use zingerone (ZN) to attract both ME-sensitive and RK-sensitive males. ZN was found as a specific male attractant for B. jarvisi. A specific attractant for the solanaceous fruit fly, B. latifrons, was characterized as 3-hydroxy-α-ionone from a specific variety of eggplants. Several 3-oxygenated α-ionone analogs were developed as monitoring agents for B. latifrons in Okinawa, Japan. Furthermore, wild Bactrocera males that have fed on attractants from certain plant sources selectively accumulate/biotransform compounds in the rectal gland possibly for sex pheromone to attract females. A sesquiterpene, β-caryophyllene, was identified from the rectal gland in wild B. correcta males. β-Caryophyllene was found to be more attractive than ME to males, and thus, potentially serves as a new monitoring agent for B. correcta. These exemplify the importance of a multidisciplinary study including behavioral, physiological, biochemical and chemoecological aspects to develop new fruit fly attractants

Conversion of 3-oxo steroids into ecdysteroids triggers molting and expression of 20E-inducible genes in Drosophila melanogaster.

Ecdysteroids, steroid hormones in insects, coordinate major developmental transitions. During postembryonic development, ecdysone is biosynthesized from dietary cholesterol in the prothoracic gland (PG). Despite extensive studies, the initial conversion process, the so-called "Black Box", has not been characterized. A cytochrome P450 enzyme, Spookier (Spok), is speculated as a rate limiting enzyme in the Black Box during larval-pupal transitions in Drosophila melanogaster. RNAi mediated knockdown of spok expression in the PG results in arrest of molting. Because the developmental arrest can be rescued by application of an appropriate intermediate, we examined potential activities of candidate intermediates in the RNAi-treated larvae. We found that two 3-oxo steroids, cholesta-4, 7-diene-3, 6-dione-14α-ol (Δ(4)-diketol) and 5β [H]cholesta-7-ene-3, 6-dione-14α-ol (diketol), triggered molting of the RNAi-treated larvae. We also detected an enhancement of the amounts of ecdysteroids in the RNAi-treated larvae by feeding the Δ(4)-diketol or diketol, indicating that the dietary 3-oxo steroids were incorporated and converted into ecdysteroids in vivo. Furthermore, 20-hydroxyecdysone inducible genes were induced in the RNAi-treated larvae by feeding the Δ(4)-diketol or diketol. These results indicate that Δ(4)-diketol and diketol are components of the ecdysteroid biosynthetic pathway and lie downstream of a step catalyzed by Spok

Floral synomone diversification of sibling Bulbophyllum species (Orchidaceae) in attracting fruit fly pollinators

Floral scent is one of the crucial cues to attract specific groups of insect pollinators in angiosperms. We examined the semiochemical diversity in the interactions between “fruit fly orchids” and their pollinator fruit fly species in two genera, Bactrocera and Zeugodacus (Tephritidae: Diptera). Two known attractants for the Dacini fruit flies, methyl eugenol (ME) and raspberry ketone (RK), have been identified from the Bulbophyllum orchids. Additionally, zingerone (ZN), with a hybrid chemical structure between ME and RK, and attracts both ME- and RK-sensitive fly species, was also identified. Male flies utilize the floral scent as sex pheromone precursor or components to attract conspecific females and gain mating advantage. We analyzed the floral components of two sibling orchids, Bulbophyllum macranthum collected from Southeast-Asian countries and Bu. praetervisum collected from Sabah, Malaysia. For Bu. macranthum, the major floral component from Malaysia and Thailand was identified as ZN; whereas that from the Philippines was ME. For Bu. praetervisum, RK was found as the major constituent, but chemical profiles of the attractants were different among individuals, i.e. in addition to RK, ZN was also found in some of the Bu. praetervisum flowers; and one of the specimens contained ME besides RK and ZN. These differences in fruit fly-attracting floral scents shown by the sympatric chemotypes of Bu. praetervisum are contrary to that shown by the allopatric chemotypes of Bu. macranthum, demonstrating the versatility in the floral synomone biosynthetic processes. Phylogenetic analysis using chloroplast DNA shows that the Malaysia- and Thailand-chemotypes of Bu. macranthum and Bu. praetervisum belong to the same lineage, although their chemical profiles are distinctly different. This demonstrates that diversification of floral synomone may happen even within a putative orchid species in order to attract a wider community of fruit fly pollinators from different genera to maximize pollination success