Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Functional analysis of SDR112C1 associated with fenpropathrin tolerance in Tetranychus cinnabarinus (Boisduval).

Short-chain dehydrogenases/reductases (SDRs) are ubiquitously distributed across diverse organisms and play pivotal roles in the growth, as well as endogenous and exogenous metabolism of various substances, including drugs. The expression levels of SDR genes are reportedly upregulated in the fenpropathrin (FEN)-resistant (FeR) strain of Tetranychus cinnabarinus. However, the functions of these SDR genes in acaricide tolerance remain elusive. In this study, the activity of SDRs was found to be significantly higher (2.26-fold) in the FeR strain compared to the susceptible strain (SS) of T. cinnabarinus. A specific upregulated SDR gene, named SDR112C1, exhibited significant overexpression (3.13-fold) in the FeR population compared with that in the SS population. Furthermore, the expression of SDR112C1 showed a significant increase in the response to FEN induction. Additionally, knockdown of the SDR112C1 gene resulted in decreased SDR activity and reduced mite viability against FEN. Importantly, heterologous expression and in vitro incubation assays confirmed that recombinant SDR112C1 could effectively deplete FEN. Moreover, the overexpression of the SDR112C1 gene in Drosophila melanogaster significantly decreased the toxicity of FEN to transgenic fruit flies. These findings suggest that the overexpression of SDR SDR112C1 is a crucial factor contributing to FEN tolerance in T. cinnabarinus. This discovery not only enhances our understanding of SDR-mediated acaricide tolerance but also introduces a new family of detoxification enzymes to consider in practice, beyond cytochrome P450s, carboxyl/choline esterases and glutathione S-transferases.

Differential pheromone profile as a contributor to premating isolation between two sympatric sibling fruit fly species.

Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) are sibling fruit fly species that are sympatric over much of their ranges. Premating isolation of these close relatives is thought to be maintained in part by allochrony-mating activity in B. tryoni peaks at dusk, whereas in B. neohumeralis, it peaks earlier in the day. To ascertain whether differences in pheromone composition may also contribute to premating isolation between them, this study used solid-phase microextraction and gas chromatography-mass spectrometry to characterize the rectal gland volatiles of a recently collected and a more domesticated strain of each species. These glands are typical production sites and reservoirs of pheromones in bactrocerans. A total of 120 peaks were detected and 50 were identified. Differences were found in the composition of the rectal gland emissions between the sexes, species, and recently collected versus domesticated strains of each species. The compositional variation included several presence/absence and many quantitative differences. Species and strain differences in males included several relatively small alcohols, esters, and aliphatic amides. Species and strain differences in females also included some of the amides but additionally involved many fatty acid esters and 3 spiroacetals. While the strain differences indicate there is also heritable variation in rectal gland emissions within each species, the species differences imply that compositional differences in pheromones emitted from rectal glands could contribute to the premating isolation between B. tryoni and B. neohumeralis. The changes during domestication could also have significant implications for the efficacy of Sterile Insect Technique control programs.

Salt concentration in substrate modulates the composition of bacterial and yeast microbiomes of Drosophila melanogaster.

Aim: Microbiomes influence the physiology and behavior of multicellular organisms and contribute to their adaptation to changing environmental conditions. However, yeast and bacterial microbiota have usually been studied separately; therefore, the interaction between bacterial and yeast communities in the gut of Drosophila melanogaster (D. melanogaster) is often overlooked. In this study, we investigate the correlation between bacterial and yeast communities in the gut of D. melanogaster. Methods: We studied the shifts in the joint microbiome of Drosophila melanogaster, encompassing both yeasts and bacteria, during adaptation to substrate with varying salt concentrations (0%, 2%, 4%, and 7%) using plating for both yeasts and bacteria and NGS-sequencing of variable 16S rRNA gene regions for bacteria. Results: The microbiome of flies and their substrates was gradually altered at moderate NaCl concentrations (2% and 4% compared with the 0% control) and completely transformed at high salt concentrations (7%). The relative abundance of Acetobacter, potentially beneficial to D. melanogaster, decreased as NaCl concentration increased, whereas the relative abundance of the more halotolerant lactobacilli first increased, peaking at 4% NaCl, and then declined dramatically at 7%. At this salinity level, potentially pathogenic bacteria of the genera Leuconostoc and Providencia were dominant. The yeast microbiome of D. melanogaster also undergoes significant changes with an increase in salt concentration in the substrate. The total yeast abundance undergoes nonlinear changes: it is lowest at 0% salt concentration and highest at 2%-4%. At a 7% concentration, the yeast abundance in flies and their substrate is lower than at 2%-4% but significantly higher than at 0%. Conclusions: The abundance and diversity of bacteria that are potentially beneficial to the flies decreased, while the proportion of potential pathogens, Leuconostoc and Providencia, increased with an increase in salt concentration in the substrate. In samples with a relatively high abundance and/or diversity of yeasts, the corresponding indicators for bacteria were often lowered, and vice versa. This may be due to the greater halotolerance of yeasts compared to bacteria and may also indicate antagonism between these groups of microorganisms.

Shaping the Microbial Landscape: Parasitoid-Driven Modifications of Bactrocera dorsalis Microbiota.

Koinobiont endoparasitoids regulate the physiology of their hosts through altering host immuno-metabolic responses, processes which function in tandem to shape the composition of the microbiota of these hosts. Here, we employed 16S rRNA and ITS amplicon sequencing to investigate whether parasitization by the parasitoid wasps, Diachasmimorpha longicaudata (Ashmaed) (Hymenoptera: Braconidae) and Psyttalia cosyrae (Wilkinson) (Hymenoptera: Braconidae), induces gut dysbiosis and differentially alter the gut microbial (bacteria and fungi) communities of an important horticultural pest, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). We further investigated the composition of bacterial communities of adult D. longicaudata and P. cosyrae to ascertain whether the adult parasitoids and parasitized host larvae share microbial taxa through transmission. We demonstrated that parasitism by D. longicaudata induced significant gut perturbations, resulting in the colonization and increased relative abundance of pathogenic gut bacteria. Some pathogenic bacteria like Stenotrophomonas and Morganella were detected in both the guts of D. longicaudata-parasitized B. dorsalis larvae and adult D. longicaudata wasps, suggesting a horizontal transfer of microbes from the parasitoid to the host. The bacterial community of P. cosyrae adult wasps was dominated by Arsenophonus nasoniae, whereas that of D. longicaudata adults was dominated by Paucibater spp. and Pseudomonas spp. Parasitization by either parasitoid wasp was associated with an overall reduction in fungal diversity and evenness. These findings indicate that unlike P. cosyrae which is avirulent to B. dorsalis, parasitization by D. longicaudata induces shifts in the gut bacteriome of B. dorsalis larvae to a pathobiont-dominated community. This mechanism possibly enhances its virulence against the pest, further supporting its candidacy as an effective biocontrol agent of this frugivorous tephritid fruit fly pest.

Dietary sucrose determines the regulatory activity of lithium on gene expression and lifespan in Drosophila melanogaster.

The amount of dietary sugars and the administration of lithium both impact the lifespan of the fruit fly Drosophila melanogaster. It is noteworthy that lithium is attributed with insulin-like activity as it stimulates protein kinase B/Akt and suppresses the activity of glycogen synthase kinase-3 (GSK-3). However, its interaction with dietary sugar has largely remained unexplored. Therefore, we investigated the effects of lithium supplementation on known lithium-sensitive parameters in fruit flies, such as lifespan, body composition, GSK-3 phosphorylation, and the transcriptome, while varying the dietary sugar concentration. For all these parameters, we observed that the efficacy of lithium was significantly influenced by the sucrose content in the diet. Overall, we found that lithium was most effective in enhancing longevity and altering body composition when added to a low-sucrose diet. Whole-body RNA sequencing revealed a remarkably similar transcriptional response when either increasing dietary sucrose from 1% to 10% or adding 1 mM LiCl to a 1% sucrose diet, characterized by a substantial overlap of nearly 500 differentially expressed genes. Hence, dietary sugar supply is suggested as a key factor in understanding lithium bioactivity, which could hold relevance for its therapeutic applications.

Forecasting the Expansion of Bactrocera tsuneonis (Miyake) (Diptera: Tephritidae) in China Using the MaxEnt Model.

The invasive pest, Bactrocera tsuneonis (Miyake), has become a significant threat to China's citrus industry. Predicting the area of potentially suitable habitats for B. tsuneonis is essential for optimizing pest control strategies that mitigate its impact on the citrus industry. Here, existing distribution data for B. tsuneonis, as well as current climate data and projections for four future periods (2021-2040, 2041-2060, 2061-2080, and 2081-2100) from the Coupled Model Intercomparison Project Phase 6 (CMIP6) were obtained. The distribution of B. tsuneonis under current and different climate change scenarios in China was predicted using the optimized MaxEnt model, ArcGIS, and the ENMeval data package. Model accuracy was assessed using ROC curves, and the primary environmental factors influencing the distribution of the pest were identified based on the percent contribution. When the regularization multiplier (RM) was set to 1.5 and the feature combination (FC) was set to LQH, a model with lower complexity was obtained. Under these parameter settings, the mean training AUC was 0.9916, and the mean testing AUC was 0.9854, indicating high predictive performance. The most influential environmental variables limiting the distribution of B. tsuneonis were the Precipitation of Warmest Quarter (Bio18) and Temperature Seasonality (standard deviation ×100) (Bio4). Under current climatic conditions, potentially suitable habitat for B. tsuneonis in China covered an area of 215.9 × 104 km2, accounting for 22.49% of the country's land area. Potentially suitable habitat was primarily concentrated in Central China, South China, and East China. However, under future climatic projections, the area of suitable habitat for B. tsuneonis exhibited varying degrees of expansion. Furthermore, the centroid of the total suitable habitat for this pest gradually shifted westward and northward. These findings suggest that B. tsuneonis will spread to northern and western regions of China under future climate changes. The results of our study indicate that climate change will have a major effect on the invasion of B. tsuneonis and have implications for the development of strategies to control the spread of B. tsuneonis in China.

Drosophila melanogaster Limostatin and Its Human Ortholog Promote West Nile Virus Infection.

The arbovirus West Nile virus (WNV) is a danger to global health. Spread primarily by mosquitoes, WNV causes about 2000 cases per year in the United States. The natural mosquito immune response controls viral replication so that the host survives but can still transmit the virus. Using the genetically malleable Drosophila melanogaster model, we previously dissected innate immune pathways used to control WNV infection. Specifically, we showed that insulin/IGF-1 signaling (IIS) activates a JAK/STAT-mediated immune response that reduces WNV. However, how factors that regulate IIS in insects control infection has not been identified. D. melanogaster Limostatin (Lst) encodes a peptide hormone that suppresses insulin secretion. Its mammalian ortholog, Neuromedin U (NMU), is a peptide that regulates the production and secretion of insulin from pancreatic beta cells. In this study, we used D. melanogaster and human cell culture models to investigate the roles of these insulin regulators in immune signaling. We found that D. melanogaster Lst mutants, which have elevated insulin-like peptide expression, are less susceptible to WNV infection. Increased levels of insulin-like peptides in these flies result in upregulated JAK/STAT activity, leading to protection from infection. Treatment of human cells with the insulin regulator NMU results in increased WNV replication. Further investigation of methods to target Lst in mosquitoes or NMU in mammals can improve vector control methods and may lead to improved therapeutics for human and animal infection.

Comparative Responses of Two Major Cucurbit Pests, Zeugodacus cucurbitae and Zeugodacus tau to Phenylbutanoid Male Lures.

Zeugodacus cucurbitae and Z. tau are two major fruit fly pests of cucurbitaceous plants in the tropical and subtropical regions. The former species has a broader host range and wider world distribution than the latter. With global climate change, Z. tau shows great potential for geographical expansion with several invasion records in recent years. Males of both species are attracted to cue lure (CL) (and raspberry ketone (RK), a deacetyl derivative of CL), a common male lure used in fruit fly population detection, monitoring and control programs. Males of both species are also known to respond to zingerone (ZN), which are produced by some rainforest orchids. Previous studies have shown that fruit fly-male lure interactions are unique to species and lure types, and significantly impact the success of a lure-based fruit fly control program. We seek to compare the attraction of Z. cucurbitae and Z. tau males to CL, RK and ZN via Probit behavioral assays. Our results showed that CL is more attractive to Z. cucurbitae and Z. tau males than RK, while ZN is a poor lure for both species. Attraction Z. tau to CL is slightly lower than Z. cucurbitae, but the former is at least 1.71 times less attractive to RK than the latter. Together with published information on species' sexual development, our current study indicates a lure-based control program via male annihilation technique for Z. tau will be more challenging than Z. cucurbitae and should incorporate other integrated pest management strategies for a desirable outcome.

Insecticidal effect of new synthesized chalcone derivatives on Caribbean fruit fly, Anastrepha suspensa.

In this present study, new chalcone derivatives were synthesized from 4-aminoacetophenone, which were confirmed by spectroscopic methods. The toxic risks of chalcones to humans and the environment were investigated by a web-based platform called ADMETlab. With this program, the possible toxic effects of the compounds on liver, respiratory system, and eyes were evaluated. For the topical insecticidal activity, adult female Caribbean fruit fly, Anastrepha suspensa, was targeted. Results of the toxicity tests showed that chalcone derivatives are effective against female A. suspensa. Among the synthesized chalcones, 1-(4-cinnamoylphenyl)-3-(p-tolyl)urea (2) exhibited the greatest insecticidal activity, resulting in 73 % mortality at 100 µg/fly after 24 h, whereas other derivatives showed less than 30 % mortality. Our results demonstrate that insecticidal activity may be modulated by the presence of a certain phenyl ring in the structure of derivative 2 and, therefore, has potential for design of efficient chemicals for tephritid fruit fly management.

Distribution and host ranges of Ceratitis rosa and Ceratitis quilicii (Diptera: Tephritidae) in South Africa.

Two fruit fly (Diptera: Tephritidae) species of economic importance: Ceratitis rosa Karsch and Ceratitis quilicii De Meyer, Mwatawala & Virgilio are present in South Africa. The two species were considered as one species prior to 2016, but were subsequently separated. In this study, the distribution and abundance of the two species were quantified in seven provinces in South Africa through trapping with Enriched Ginger Oil as an attractant. Trapping was conducted over three seasons across two years (2020 and 2021): late summer, autumn-winter, and spring-early summer. Host ranges of the two species were investigated by fruit sampling in and outside of trapping sites. Ceratitis quilicii was more widely distributed than C. rosa with the latter being recorded in only three north-eastern provinces. There were geographical limits for both species with no records of them in Northern Cape Province. Catches of C. quilicii were higher in summer with average temperatures varying from 15 to 27°C while for C. rosa, catches remained low and consistent between seasons. Ceratitis quilicii catches decreased at lower rates than those of C. rosa at temperatures below 15°C. The two species were reared from 13 plant species from nine families. Four of these hosts were infested by both C. quilicii and C. rosa in the same province where they occurred. Preferred hosts of the two species belonged to the Myrtaceae family. The characterisation of the distribution, abundance and host ranges of these pests will provide a baseline for pest status determination and implementation of management actions.

The genome sequence of the drosophilid fruit fly, Drosophila phalerata (Meigen, 1830).

We present a genome assembly from an individual male Drosophila phalerata (drosophilid fruit fly, Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 223.9 megabases in span. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 16.14 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,973 protein coding genes.

Among- and Within-Individual Variance in Metabolic Thermal Reaction Norms.

AbstractIn ectotherms, temperature has a strong effect on metabolic rate (MR), yet the extent to which the thermal sensitivity of MR varies among versus within individuals is largely unknown. This is of interest because significant among-individual variation is a prerequisite for the evolution of metabolic thermal sensitivity. Here, we estimated the repeatability (R) of the thermal sensitivity of MR in individual virgin, adult male Drosophila melanogaster (N=316) by taking repeated overnight measures of their MRs at two temperatures (~24°C and ~27°C). At the population level, thermal sensitivity decreased with locomotor activity, and older individuals showed a higher thermal sensitivity of MR than younger individuals. Taking these effects (and body mass) into account, we detected significant repeatability in both the centered intercept (Rint=0.52±0.04) and the slope (Rslp=0.21±0.07) of the metabolic thermal reaction norms, which respectively represent average MR and thermal sensitivity of MR. Furthermore, individuals with a higher overall MR also displayed greater increases in MR as temperature increased from ~24°C to ~27°C (rind=0.32±0.14). Average MR and thermal sensitivity of MR were also positively correlated within individuals (re=0.15±0.07). Our study represents a point of departure for future larger studies, in which more complex protocols (e.g., wider temperature range, breeding design) can be applied to quantify the causal components of variation in thermal sensitivity that are needed to make accurate predictions of adaptive responses to global warming.

Two Old Wild-Type Strains of Drosophila melanogaster Can Serve as an Animal Model of Faster and Slower Aging Processes.

BACKGROUND: Drosophila melanogaster provides a powerful platform to study the physiology and genetics of aging, i.e., the mechanisms underpinnings healthy aging, age-associated disorders, and acceleration of the aging process under adverse environmental conditions. Here, we tested the responses of daily rhythms to age-accelerated factors in two wild-type laboratory-adapted strains, Canton-S and Harwich. METHODS: On the example of the 24 h patterns of locomotor activity and sleep, we documented the responses of these two strains to such factors as aging, high temperature, carbohydrate diet, and diet with different doses of caffeine-benzoate sodium. RESULTS: The strains demonstrated differential responses to these factors. Moreover, compared to Canton-S, Harwich showed a reduced locomotor activity, larger amount of sleep, faster rate of development, smaller body weight, lower concentrations of main sugars, lower fecundity, and shorter lifespan. CONCLUSIONS: It might be recommended to use at least two strains, one with a relatively fast and another with a relatively slow aging process, for the experimental elaboration of relationships between genes, environment, behavior, physiology, and health.

Overexpression of an Integument Esterase Gene LbEST-inte4 Infers the Malathion Detoxification in Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Liposcelis bostrychophila, commonly known as booklouse, is an important stored-product pest worldwide. Studies have demonstrated that booklices have developed resistance to several insecticides. In this study, an integument esterase gene, LbEST-inte4, with upregulated expression, was characterized in L. bostrychophila. Knockdown of LbEST-inte4 resulted in a substantial increase in the booklice susceptibility to malathion. Overexpression of LbEST-inte4 in Drosophila melanogaster significantly enhanced its malathion tolerance. Molecular modeling and docking analysis suggested potential interactions between LbEST-inte4 and malathion. When overexpressed LbEST-inte4 in Sf9 cells, a notable elevation in esterase activity and malathion tolerance was observed. HPLC analysis indicated that the LbEST-inte4 enzyme could effectively degrade malathion. Taken together, the upregulated LbEST-inte4 appears to contribute to malathion tolerance in L. bostrychophila by facilitating the depletion of malathion. This study elucidates the molecular mechanism underlying malathion detoxification and provides the foundations for the development of effective prevention and control measures against psocids.

Curcumin-mediated alleviation of dextran-induced leaky gut in Drosophila melanogaster.

Aging is commonly characterized by a decline in the physiological functioning of the body organs, with one hallmark being the impairment of intestinal function, leading to increased intestinal permeability known as leaky gut. The aim of this study was to investigate the potential of curcumin to prevent the development of leaky gut in Drosophila melanogaster utilizing the smurf fly method. In this study, flies aged 3-5 days underwent a 10-day dextran sulfate sodium (DSS) treatment to induce intestinal permeability, followed by a smurf assay using brilliant blue dye and locomotor testing the next day. Flies displaying the smurf phenotype were divided into four groups: untreated control and curcumin-treated (10 µM, 50 µM, and 250 µM). After 21 days of treatment, flies were reassessed for the smurf phenotype and underwent locomotor testing. On day 23, flies were subjected to RT-qPCR analysis. By inducing increased intestinal permeability through the administration of DSS, a higher proportion of flies exhibiting the smurf phenotype and a reduced survival rate in the DSS-treated group were observed. Such phenotypes were reversed, decreased number of flies displaying the smurf phenotype and improved fly survival, upon the incorporation of curcumin in the fly food at concentrations of 10, 50, and 250 µM. Subsequent molecular analysis revealed upregulated expression of sod1, cat, and pepck genes, while no significant changes were observed in the expression of sod2, indy, and srl genes following treatment with curcumin at high concentration. Overall, our findings provide insight into the potential effect of curcumin to alleviate the phenotypical features associated with DSS-induced leaky gut, possibly via the selective regulation of aging-related genes.

Oviposition-Deterrent Effect of a High-Quality Natural Zeolite on the Olive Fruit Fly Bactrocera oleae, under Different Conditions of Temperature and Relative Humidity.

In recent years, the number of available chemical pesticides has been dramatically reduced, urging the need for the discovery of alternatives to chemical pesticide products such as, among others, natural zeolites (zeolitic rocks). We determined the mineralogical and chemical composition of a specific and continuous layer of zeolitic rock sample (ZeotP) from Petrota, Evros, Greece, and evaluated its oviposition-deterrent effect on the olive fruit fly Bactrocera oleae Gmelin (Diptera: Terphritidae). The tested natural zeolite contained 70 wt. % clinoptilolite, 18 wt. % amorphous material, 7 wt. % feldspars, 4 wt. % cristobalite, and 1 wt. % quartz. We tested the oviposition-deterrent effect of ZeotP mixed or not with an emulsifier adjuvant, NU-FILM-P®, in water and applied it to the surface of olive fruits. The ZeotP oviposition-deterrent effect on the olive fly was very high under a series of tested temperatures (17 °C, 20 °C, 25 °C, and 30 °C) and RHs (23%, 33%, 55%, 75%, and 94%). In addition, the ZeotP residual deterrent effect after equable water spraying was high, like the respective effect of the pyrethroid insecticide Decis® (deltamethrin). Our results may contribute to the effective control of the olive fruit fly using an alternative to chemical pesticides: natural zeolite (zeolitic rocks) products.

Drosophila melanogaster as a model organism for diabetes II treatment by the ethyl acetate fraction of Atriplex halimus L.

Type 2 diabetes (T2D) is the most common metabolic disorder. The undesirable effects of synthetic drugs demand a search for safe antidiabetic agents. This study aimed to assess the antidiabetic activity of different fractions of Atriplex halimus (petroleum ether 60-80, methylene chloride, ethyl acetate, and n-butanol) using Drosophila melanogaster larvae. Titers of total glucose and trehalose, as well as larval weight, were measured and compared with those of control and diabetic larvae. The expression of Drosophila insulin-like peptides (DILP2 and DILP3) and adipokinetic hormone (AKH) was evaluated. The results revealed a significant increase in total glucose, trehalose, and a decrease in body weight in the larvae fed a high-sugar diet compared with those in the control. When larvae fed diets containing the tested fractions, the total glucose and trehalose decreased to the control level, and the body weight increased. DILP2, DILP3, and AKH exhibited significant decreases upon treatment with A. halimus ethyl acetate. Metabolomic profiling of the ethyl acetate fraction of A. halimus revealed the presence of flavonoids and flavonoid glycosides. After docking screening to predict the most powerful moiety, we discovered that flavonoid glycosides (especially eriodictyol-7-O-neohesperidoside) have a greater affinity for the pocket than the other moieties. The results indicated the therapeutic activity of the A. halimus ethyl acetate fraction against induced T2D in Drosophila larvae. The antidiabetic activity may be attributed to flavonoids, which are the main components of the A. halimus ethyl acetate fraction.

Investigating the thermal sensitivity of key enzymes involved in the energetic metabolism of three insect species.

The metabolic responses of insects to high temperatures have been linked to their mitochondrial substrate oxidation capacity. However, the mechanism behind this mitochondrial flexibility is not well understood. Here, we used three insect species with different thermal tolerances (the honey bee, Apis mellifera; the fruit fly, Drosophila melanogaster; and the potato beetle, Leptinotarsa decemlineata) to characterize the thermal sensitivity of different metabolic enzymes. Specifically, we measured activity of enzymes involved in glycolysis (hexokinase, HK; pyruvate kinase, PK; and lactate dehydrogenase, LDH), pyruvate oxidation and the tricarboxylic acid cycle (pyruvate dehydrogenase, PDH; citrate synthase, CS; malate dehydrogenase, MDH; and aspartate aminotransferase, AAT), and the electron transport system (Complex I, CI; Complex II, CII; mitochondrial glycerol-3-phosphate dehydrogenase, mG3PDH; proline dehydrogenase, ProDH; and Complex IV, CIV), as well as that of ATP synthase (CV) at 18, 24, 30, 36, 42 and 45°C. Our results show that at high temperature, all three species have significantly increased activity of enzymes linked to FADH2 oxidation, specifically CII and mG3PDH. In fruit flies and honey bees, this coincides with a significant decrease of PDH and CS activity, respectively, that would limit NADH production. This is in line with the switch from NADH-linked substrates to FADH2-linked substrates previously observed with mitochondrial oxygen consumption. Thus, we demonstrate that even though the three insect species have a different metabolic regulation, a similar response to high temperature involving CII and mG3PDH is observed, denoting the importance of these proteins for thermal tolerance in insects.

Assessment of the efficacy of Piper methysticum (Micrembryeae: Piperaceae) as a bioinsecticide, and/or spinosad combined with attractive plant volatiles for a novel lure and kill strategy against Bactrocera tryoni (Diptera: Tephritidae) in laboratory.

The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is a crop pest of global economic importance because of its wide range of hosts and its invasiveness capacities. To develop a novel integrated and sustainable crop protection, we have investigated the insecticidal properties of different varieties of kava (Piper methysticum [Frost]) extracted by two methods and the attractive effects of six plant volatiles identified from B. tryoni host plants to female, mated or not. We did not identify any significant insecticidal effect of the traditional Pacific kava plant at the tested concentrations. Among mated females, ethyl acetate compared to the no odor control elicited the highest attraction (87%, of which 60% for this odor), while ethyl butyrate was preferred compared with ethyl acetate in dual choice assays. Flies' preferences for specific odors depended on their mating status and the odor landscape they were confronted with. Combination with the commercial ingestion insecticide (Success 4: spinosad, 480 g/l, Dow AgroSciences, Valbonne, France) with the plant volatiles were tested to detect an increase in mortality related to the addition of an attractant. The 2-heptanone slightly showed a tend to increase the attractiveness of mated females within 4-6 h to the food bait, but the results were not statistically significant after 8 h. Further tests should be performed with other concentrations or mixtures of the identified host plant volatiles to develop a strong lure and kill strategy.