Raspberry ketone diet supplement reduces attraction of sterile male Queensland fruit fly to cuelure by altering expression of chemoreceptor genes.

Sterile male Queensland fruit fly, Bactrocera tryoni (Froggatt), fed as immature adults on the plant compound raspberry ketone (RK), show a reduced attraction to cuelure, a synthetic analogue of RK used as an attractant in Male Annihilation Technique. We hypothesized the reduced attraction of RK-fed adult males to cuelure may be a consequence of altered expression of chemoreception genes. A Y-tube olfactometer assay with RK-fed and RK-unfed sterile B. tryoni males tested the subsequent behavioural response to cuelure. Behavioral assays confirmed a significant decrease in attraction of RK-fed sterile males to cuelure. RK-fed, non-responders (to cue-lure) and RK-unfed, responders (to cue-lure) males were sampled and gene expression compared by de novo RNA-seq analysis. A total of 269 genes in fly heads were differentially expressed between replicated groups of RK-fed, cuelure non-responders and RK-unfed, cuelure responders. Among them, 218 genes including 4 chemoreceptor genes were up regulated and 51 genes were down regulated in RK-fed, cuelure non-responders. De novo assembly generated many genes with unknown functions and no significant BLAST hits to homologues in other species. The enriched and suppressed genes reported here, shed light on the transcriptional changes that affect the dynamics of insect responses to chemical stimuli.

Comparative Proteomic Profiling between Each of Two Consecutive Developmental Stages of the Solanum Fruit Fly, Bactrocera latifrons (Hendel).

The Solanum fruit fly, Bactrocera latifrons (Hendel), has a complex life cycle including multiple stages (egg, larva, pupa, and adult). Understanding the details of "what", "when", "where", "why", and "how" many hundred thousand proteins operate in this insect, interact, and express between each two consecutive developmental stages at molecular level not only can expand our knowledge, but also lead to the development of novel fruit fly control techniques. We tried to find what, when, and where in this study. Why and how will be presented in upcoming papers. We conducted a proteome profiling using 2-D gel electrophoresis and mass spectrometry. Samples of 3-day-old eggs, 1- and 10-day-old larvae, 1- and 10-day-old pupae, 1- and 9-day-old females and males of B. latifrons were used. A custom peptide database, derived from the de novo B. latifrons whole genome assembly was used for peptide identification. Differentially expressed proteins (DEPs) with significant fold expression and protein functions between two consecutive developmental stages were identified, annotated, described, and listed in gel images and/or charts. With this foundational information, we are not only providing valuable information, but also any impacts due to the biotic or abiotic environmental factors can be identified and manipulated, and lead to further research on gene editing and biomarker discovery.

Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.

Field trials of solid triple lure (trimedlure, methyl eugenol, raspberry ketone, and DDVP) dispensers for detection and male annihilation of Ceratitis capitata, Bactrocera dorsalis, and Bactrocera cucurbitae (Diptera: Tephritidae) in Hawaii.

Solid Mallet TMR (trimedlure [TML], methyl eugenol [ME], raspberry ketone [RK]) wafers and Mallet CMR (ceralure, ME, RK, benzyl acetate) wafers impregnated with DDVP (2,2-dichlorovinyl dimethyl phosphate) insecticide were measured in traps as potential detection and male annihilation technique (MAT) devices. Comparisons were made with 1) liquid lure and insecticide formulations, 2) solid cones and plugs with an insecticidal strip, and 3) solid single and double lure wafers with DDVP for captures of Mediterranean fruit fly, Ceratitis capitata (Wiedemann); oriental fruit fly, Bactrocera dorsalis Hendel; and melon fly, B. cucurbitae Coquillett. Bucket and Jackson traps were tested in a coffee plantation near Eleele, Kauai Island, HI (trials at high populations) and avocado orchards near Kona, HI Island, HI (trials at low populations). Captures of all three species with Mallet TMR were not different from Mallet CMR; therefore, subsequent experiments did not include Mallet CMR because of higher production costs. In MAT trials near Eleele, HI captures in AWPM traps with Mallet TMR wafers were equal to any other solid lure (single or double) except the Mallet ME wafer. In survey trials near Kona, captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in FL and CA. A solid Mallet TMR wafer is safer, more convenient to handle, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and MAT programs in Hawaii.

Larval dietary wheat germ oil influences age-specific protein expression in adults of the oriental fruit fly.

Changes in essential dietary components alter global gene expression patterns in animals. We reported on a proteomics study designed to identify molecular markers of deficiencies in culture media developed for the oriental fruit fly, Bactrocera dorsalis. In that study, we found significant changes in expression of 70 proteins in adults of larvae reared on media lacking wheat germ oil (WGO), compared to media supplemented with WGO. Of these, a gene encoding an insect chitin-binding protein was expressed at about 120-fold higher levels in adult males reared on media supplemented with WGO. We inferred it may be feasible to develop the gene as a molecular marker of dietary lipid deficiency. The work was focused, however, on analysis of 11 day old adults. We have no information on expression of the chitin-binding protein, nor on any other proteins at other adult ages. In this paper we address the idea that the whole animal proteome changes dynamically with age. We reared separate groups of fruit fly larvae on media with and without WGO supplementation and analyzed protein expression in adult males and females age 0, 4, 8 and 12 days old using 2D electrophoresis. Gel densitometry revealed significant increases (by >2-fold) and decreases (by >50%) in expression levels of 29 proteins in females and 10 in males. We identified these proteins by mass spectrometry on MALDI TOF/TOF and bioinformatic analyses of the protein sequences. Two proteins, peroxiredoxin (26-fold increase) and vitellogenin 1 (15-fold increase) increased in expression in day 8 females. The key finding is that most changes in protein expression occurred in day 8 females. We infer that the fruit fly proteome changes with adult age. The natural changes in proteome with adult age is a crucial aspect of developing these and other proteins into molecular markers of lipid deficiency in fruit flies and possibly other insect species.

Dietary wheat germ oil influences gene expression in larvae and eggs of the oriental fruit fly.

Changes in animal nutrition, particularly essential dietary components, alter global gene expression patterns. Our goal is to identify molecular markers that serve as early indicators of the quality of insect culture media. Markers of deficient culture media will increase the efficiency of developing optimal systems for mass rearing beneficial insects and some pest species because decisions on culture media quality can be made without waiting through one or several life cycles. The objective of our current study is to discover molecular markers of essential dietary lipid deficiency in the oriental fruit fly, Bactrocera dorsalis. We reared groups of fruit flies separately on media either devoid of or supplemented with wheat germ oil (WGO) and analyzed gene expression in third instar larvae and F(1) eggs using 2D electrophoresis. Gel densitometry revealed significant changes in expression levels of genes encoding eight proteins in larvae and 22 proteins in eggs. We identified these proteins by using mass spectrometry (MALDI TOF/TOF) and bioinformatic analyses of the protein sequences. Among these, we identified one gene encoding the receptor of activated C Kinase 1 (RACK1) that increased in expression by 6.8-fold in eggs from adults that were reared as larvae on media supplemented with WGO. RACK1 is an essential component of at least three intracellular signal transduction pathways, making it a good molecular marker candidate of lipid deficiency in fruit flies and possibly many other insect species.

Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon fly Bactrocera cucurbitae: effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast.

The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments known to enhance mating success in the male melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), supporting their use in mass rearing protocols for sterile males in the context of sterile insect technique (SIT) programmes. The objective of the present laboratory study was to investigate the effect of methoprene application and diet supplementation with hydrolyzed yeast (protein) on the turnover of body lipids and protein to confirm the feasibility of their application in melon fly SIT mass-rearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males were exposed to one of the following treatments: (1) topical application of methoprene and access to diet including protein (M+P+); (2) only diet including protein (M-P+); (3) only methoprene (M+P-) and (4) untreated, only sugar-fed, control males (M-P-). Total body carbon (TBC) and total body nitrogen (TBN) of flies were measured at regular intervals from emergence to 35 days of age for each of the different treatments. Nitrogen assimilation and turnover in the flies were measured using stable isotope ((15)N) dilution techniques. Hydrolyzed yeast incorporation into the diet significantly increased male body weight, TBC and TBN as compared to sugar-fed males. Females had significantly higher body weight, TBC and TBN as compared to all males. TBC and TBN showed age-dependent changes, increasing until the age of sexual maturity and decreasing afterwards in both sexes. Methoprene treatment did not significantly affect TBC or TBN. The progressive increase with age of TBC suggests that lipogenesis occurs in adult male B. cucurbitae, as is the case in other tephritids. Stable isotope dilution was shown to be an effective method for determining N uptake in B. cucurbitae. This technique was used to show that sugar-fed males rely solely on larval N reserves and that the N uptake rate in males with access to diet including hydrolyzed yeast was higher shortly after emergence and then stabilized. The implications of the results for SIT applications are discussed.