Optimization of Brewer's Yeast Quantity in Liquid and Gel Larval Diets for the Mediterranean Fruit Fly.

Several artificial larval diets have been developed, evaluated and used for mass-rearing of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Teprhitidae). There are several efforts to reduce the cost of rearing and optimize the quality of the produced sterile males that are destined for release in sterile insect release programs. Survival, growth, longevity and reproductive capacity of sterile males are strongly connected with the most expensive ingredient, the brewer's yeast (protein), in the larval diet. The current study focused on settling the optimal content of brewer's yeast in a liquid diet and a gel diet. Egg hatch rates, developmental duration of immatures, pupation rate, pupae and adult survival were recorded as indicators of quantity and quality of the produced adults. Egg hatch was higher and larval developmental duration longer in the gel diet. In contrast to the liquid diet, an increase in brewer's yeast concentration was correlated with increased pupation rate and pupae survival in the gel diet. Reducing brewer's yeast up to 50% of its initial quantity had no significant effect on the survival of the emerging adults regardless of the diet type. Our findings may contribute to the production of low-cost and effective diets for use in mass-rearing facilities of medflies.

Effect of thermal acclimation on the tolerance of the peach fruit fly (Bactrocera zonata: Tephritidae) to heat and cold stress.

Understanding the thermal biology of insects is of increasing importance for predicting their geographic distribution, particularly in light of current and future global temperature increases. Within the limits set by genetic makeup, thermal tolerance is affected by the physiological conditioning of individuals (e.g., through acclimation). Considering this phenotypic plasticity may add to accurately estimating changes to the distribution of insects under a changing climate. We studied the effect of thermal acclimation on cold and heat tolerance of the peach fruit fly (Bactrocera zonata) - an invasive, polyphagous pest that is currently expanding through Africa and the Middle East. Females and males were acclimated at 20, 25 and 30 °C for up to 19 days following adult emergence. The critical thermal minimum (CTmin) and maximum (CTmax) were subsequently recorded as well adult survival following acute exposure to chilling (0 or -3 °C for 2 h). Additionally, we determined the survival of pupae subjected for 2 h to temperatures ranging from -12 °C to 5 °C. We demonstrate that acclimation at 30 °C resulted in significantly higher CTmax and CTmin values (higher heat resistance and lower cold resistance, respectively). Additionally, adult recovery following exposure to -3 °C was significantly reduced following acclimation at 30 °C, and this effect was significantly higher for females. Pupal mortality increased with the decrease in temperature, reaching LT50 and LT95 values following exposure to -0.32 °C and -6.88 °C, respectively. Finally, we found that the survival of pupae subjected to 0 and 2 °C steadily increased with pupal age. Our findings substantiate a physiological foundation for understanding the current geographic range of B. zonata. We assume that acclimation at 30 °C affected the thermal tolerance of the flies partly through modulating feeding and metabolism. Tolerance to chilling during the pupal stage probably changed according to temperature-sensitive processes occurring during metamorphosis, rendering younger pupae more sensitive to chilling.

Acute cold stress and supercooling capacity of Mediterranean fruit fly populations across the Northern Hemisphere (Middle East and Europe).

The Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), holds an impressive record of successful invasion events promoted by globalization in fruit trade and human mobility. In addition, C. capitata is gradually expanding its geographic distribution to cooler temperate areas of the Northern Hemisphere. Cold tolerance of C. capitata seems to be a crucial feature that promotes population establishment and hence invasion success. To elucidate the interplay between the invasion process in the northern hemisphere and cold tolerance of geographically isolated populations of C. capitata, we determined (a) the response to acute cold stress survival of adults, and (b) the supercooling capacity (SCP) of immature stages and adults. To assess the phenotypic plasticity in these populations, the effect of acclimation to low temperatures on acute cold stress survival in adults was also examined. The results revealed that survival after acute cold stress was positively related to low temperature acclimation, except for females originating from Thessaloniki (northern Greece). Adults from the warmer environment of South Arava (Israel) were less tolerant after acute cold stress compared with those from Heraklion (Crete, Greece) and Thessaloniki. Plastic responses to cold acclimation were population specific, with the South Arava population being more plastic compared to the two Greek populations. For SCP, the results revealed that there is little to no correlation between SCP and climate variables of the areas where C. capitata populations originated. SCP was much lower than the lowest temperature individuals are likely to experience in their respective habitats. These results set the stage for asking questions regarding the evolutionary adaptive processes that facilitate range expansions of C. capitata into cooler temperate areas of Europe.

Effects of Thermal Acclimation on the Tolerance of Bactrocera zonata (Diptera: Tephritidae) to Hydric Stress.

Insects, similarly to other small terrestrial invertebrates, are particularly susceptible to climatic stress. Physiological adjustments to cope with the environment (i.e., acclimation) together with genetic makeup eventually determine the tolerance of a species to climatic extremes, and constrain its distribution. Temperature and desiccation resistance in insects are both conditioned by acclimation and may be interconnected, particularly for species inhabiting xeric environments. We determined the effect of temperature acclimation on desiccation resistance of the peach fruit fly (Bactrocera zonata, Tephritidae) - an invasive, polyphagous pest, currently spreading through both xeric and mesic environments in Africa and the Eurasian continent. Following acclimation at three constant temperatures (20, 25, and 30°C), the survival of adult flies deprived of food and water was monitored in extreme dry and humid conditions (<10 and >90% relative humidity, respectively). We found that flies acclimated at higher temperatures were significantly heavier, and contained more lipids and protein. Acclimation temperature significantly and similarly affected the survival of males and females at both high and low humidity conditions. In both cases, flies maintained at 30°C survived longer compared to 20 and 25°C - habituated counterparts. Regardless of the effect of acclimation temperature on survival, overall life expectancy was significantly shortened when flies were assayed under desiccating conditions. Additionally, our experiments indicate no significant difference in survival patterns between males and females, and that acclimation temperature had similar effects after both short (5-10 days) and long (11-20 days) acclimation periods. We conclude that acclimation at 30°C prolongs the survival of B. zonata, regardless of ambient humidity levels. Temperature probably affected survival through modulating feeding and metabolism, allowing for accumulation of larger energetic reserves, which in turn, promoted a greater ability to resist starvation, and possibly desiccation as well. Our study set the grounds for understanding the phenotypic plasticity of B. zonata from the hydric perspective, and for further evaluating the invasion potential of this pest.

New Viruses from the Ectoparasite Mite Varroa destructor Infesting Apis mellifera and Apis cerana.

Varroa destructor is an ectoparasitic mite of Asian or Eastern honeybees Apis cerana(A. cerana) which has become a serious threat to European subspecies of Western honeybees Apis mellifera (A. mellifera) within the last century. V.destructor and its vectored honeybee viruses became serious threats for colony survival. This is a short period for pathogen- and host-populations to adapt. To look for possible variation in the composition of viral populations we performed RNA metagenomic analysis of the Western honeybee subspecies A. m. ligustica, A. m.syriaca, A. m. intermissa, and A. cerana and their respective V. destructor mites. The analysis revealed two novel viruses: Varroa orthomyxovirus-1 (VOV-1) in A. mellifera and V. destructor and a Hubei like-virga virus-14 homolog in V. destructor. VOV-1 was more prevalent in V. destructor than in A. mellifera and we found evidence for viral replication in both hosts. Interestingly, we found differences in viral loads of A. cerana and their V. destructor, A. m. intermissa, and its V. destructor showed partial similarity, while A. m.ligustica and A. m.syriaca and their varroa where very similar. Deformed wing virus exhibited 82.20%, 99.20%, 97.90%, and 0.76% of total viral reads in A. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana, respectively. This is the first report of a complete segmented-single-stranded negative-sense RNA virus genome in honeybees and V. destructor mites.

Age Related Assessment of Sugar and Protein Intake of Ceratitis capitata in ad libitum Conditions and Modeling Its Relation to Reproduction.

In the inquiry on the age related dietary assessment of an organism, knowledge of the distributional patterns of food intake throughout the entire life span is very important, however, age related nutritional studies often lack robust feeding quantification methods due to their limitations in obtaining short-term food-intake measurements. In this study, we developed and standardized a capillary method allowing precise life-time measurements of food consumption by individual adult medflies, Ceratitis capitata (Diptera: Tephritidae), under laboratory conditions. Protein or sugar solutions were offered via capillaries to individual adults for a 5 h interval daily and their consumption was measured, while individuals had lifetime ad libitum access to sugar or protein, respectively, in solid form. Daily egg production was also measured. The multivariate data-set (i.e., the age-dependent variations in the amount of sugar and protein ingestion and their relation to egg production) was analyzed using event history charts and 3D interpolation models. Maximum sugar intake was recorded early in adult life; afterwards, ingestion progressively dropped. On the other hand, maximum levels of protein intake were observed at mid-ages; consumption during early and late adult ages was kept at constant levels. During the first 30 days of age, type of diet and sex significantly contributed to the observed difference in diet intake while number of laid eggs varied independently. Male and female adult longevity was differentially affected by diet: protein ingestion extended the lifespan, especially, of males. Smooth surface models revealed a significant relationship between the age dependent dietary intake and reproduction. Both sugar and protein related egg-production have a bell-shaped relationship, and the association between protein and egg-production is better described by a 3D Lorenzian function. Additionally, the proposed 3D interpolation models produced good estimates of egg production and diet intake as affected by age, providing us with a reliable multivariate analytical tool to model nutritional trends in insects, and other organisms, and their effect upon life history traits. The modeling also strengthened the knowledge that egg production is closely related to protein consumption, as suggested by the shape of the medfly reproduction-response function and its functional relationship to diet intake and age.

Resource allocation and compensation during development in holometabolous insects.

We provide an extensive review on current knowledge and future research paths on the topic of resource allocation and compensation during development in holometabolous insects, emphasizing the role of resource management during development, and how compensatory mechanisms may be acting to remediate nutritional deficiencies carried over from earlier stages of development. We first review resource allocation in "open" and "closed" developmental stages and then move on to the topic of modelling resource allocation and its trade-offs. In doing so, we review novel methodological developments such as response-surface methods and mixture experiments as well as nutritional geometry. We also dwell on the fascinating topic of compensatory physiology and behavior. We finish by discussing future research paths, among them the emerging field of nutrigenomics and gut microbiome, which will shed light into the yet poorly understood role of the symbiotic microbiota in nutrient compensation or assimilation.

Olive Fruit Fly (Bactrocera oleae) Population Dynamics in the Eastern Mediterranean: Influence of Exogenous Uncertainty on a Monophagous Frugivorous Insect.

Despite of the economic importance of the olive fly (Bactrocera oleae) and the large amount of biological and ecological studies on the insect, the factors driving its population dynamics (i.e., population persistence and regulation) had not been analytically investigated until the present study. Specifically, our study investigated the autoregressive process of the olive fly populations, and the joint role of intrinsic and extrinsic factors molding the population dynamics of the insect. Accounting for endogenous dynamics and the influences of exogenous factors such as olive grove temperature, the North Atlantic Oscillation and the presence of potential host fruit, we modeled olive fly populations in five locations in the Eastern Mediterranean region. Our models indicate that the rate of population change is mainly shaped by first and higher order non-monotonic, endogenous dynamics (i.e., density-dependent population feedback). The olive grove temperature was the main exogenous driver, while the North Atlantic Oscillation and fruit availability acted as significant exogenous factors in one of the five populations. Seasonal influences were also relevant for three of the populations. In spite of exogenous effects, the rate of population change was fairly stable along time. We propose that a special reproductive mechanism, such as reproductive quiescence, allows populations of monophagous fruit flies such as the olive fly to remain stable. Further, we discuss how weather factors could impinge constraints on the population dynamics at the local level. Particularly, local temperature dynamics could provide forecasting cues for management guidelines. Jointly, our results advocate for establishing monitoring programs and for a major focus of research on the relationship between life history traits and populations dynamics.

Conserved metallomics in two insect families evolving separately for a hundred million years.

Μetal cofactors are required for enzymatic catalysis and structural stability of many proteins. Physiological metal requirements underpin the evolution of cellular and systemic regulatory mechanisms for metal uptake, storage and excretion. Considering the role of metal biology in animal evolution, this paper asks whether metal content is conserved between different fruit flies. A similar metal homeostasis was previously observed in Drosophilidae flies cultivated on the same larval medium. Each species accumulated in the order of 200 µg iron and zinc and approximately ten-fold less manganese and copper per gram dry weight of the adult insect. In this paper, data on the metal content in fourteen species of Tephritidae, which are major agricultural pests worldwide, are presented. These fruit flies can be polyphagous (e.g., Ceratitis capitata) or strictly monophagous (e.g., Bactrocera oleae) or oligophagous (e.g., Anastrepha grandis) and were maintained in the laboratory on five distinct diets based on olive oil, carrot, wheat bran, zucchini and molasses, respectively. The data indicate that overall metal content and distribution between the Tephritidae and Drosophilidae species was similar. Reduced metal concentration was observed in B. oleae. Feeding the polyphagous C. capitata with the diet of B. oleae resulted in a significant quantitative reduction of all metals. Thus, dietary components affect metal content in some Tephritidae. Nevertheless, although the evidence suggests some fruit fly species evolved preferences in the use or storage of particular metals, no metal concentration varied in order of magnitude between these two families of Diptera that evolved independently for over 100 million years.

Analyzing diurnal and age-related pheromone emission of the olive fruit fly, Bactrocera oleae by sequential SPME-GCMS analysis.

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), uses 1,7-dioxaspiro[5.5]undecane ("olean"), produced primarily by females, as a sex pheromone. We used sequential solid phase microextraction-gas chromatography mass spectrometry (SPME-GCMS) analysis to show that female olive flies release about 1000 ng of pheromone at the onset of scotophase for several weeks, while males release about 1/100 as much during the first week after eclosion. The present research demonstrates details of employing SPME-GCMS with the partially known pheromone system of the olive fruit fly as a model for pheromone identification and diurnal release patterns in insects, especially fruit flies. The sequential SPME-GCMS method will readily allow detection and semi-quantification of semiochemicals released by insects in minute amounts throughout the diurnal cycle.

Coping with an unpredictable and stressful environment: the life history and metabolic response to variable food and host availability in a polyphagous tephritid fly.

The way energy resources are used under variable environmental conditions lies at the heart of our understanding of resource management and opportunism in many organisms. Here we sought to determine how a time-limited, synovigenic and polyphagous insect with a high reproductive-potential (Anastrephaludens), copes behaviourally and metabolically with environmental unpredictability represented by constant and variable regimes of host availability and variation in food quality. We hypothesized that an adaptive response to a windfall of nutritious food would be the rapid accumulation of energy metabolites (whole body lipids, glycogen and proteins) in the female. We also studied patterns of oogenesis as an indicator of egg-reabsorption under stressful environmental conditions. As predicted, patterns of energy metabolites were mainly driven by the quality and temporal pattern of food availability. In contrast, patterns of host availability had a lower impact upon metabolites. When given constant access to high quality nutrients, after an initial increase early in life, whole body lipids and glycogen were regulated downward to a steady-state level and somatic protein levels did not vary. In contrast, when food uncertainty was introduced, whole body lipid, glycogen and protein oscillated sharply with peaks associated with pulses of high-quality food. Production of eggs was highest when offered continuous access to hosts and high quality food. Importantly, females fully recovered their reproductive capacity when fruit became available following a period of host deprivation. With no evidence of egg resorption and high levels of egg dumping, it appears that egg dumping may favour the continuous production of eggs such that the female's reproductive tissues are ready to respond to rapid changes in the availability of hosts. Our results exemplify the capacity of insects to maximize reproduction under variable and stressful environmental conditions.

Physiological and biological patterns of a highland and a coastal population of the European cherry fruit fly during diapause.

Adult emergence of univoltine temperate insect species and its synchronization with specific host phenological stages is mainly regulated by obligatory pupal diapause. Although a few studies have investigated the factors affecting diapause intensity, little attention has been paid to the physiological alterations and metabolic regulation that take place during diapause. Here, we describe differences in diapause between a highland and a coastal Greek population of the European cherry fruit fly Rhagoletis cerasi, a major pest of sweet and sour cherries in many European countries. Pupae of both populations were exposed to the environmental conditions prevailing in the two areas and diapause termination was observed under laboratory conditions. The regulation of energetic metabolites during the long pupae stage was examined under both field and laboratory conditions. Differences in diapause intensity revealed that the two populations have adapted to the local geographical and climatic conditions and have different requirements for low temperatures to terminate diapause. The coastal population undergoes a shorter diapause and adults emerge more rapidly, especially in the highland area. The highland population failed to terminate diapause (<40% adult emergence) in the coastal area. Both populations draw on their major energetic reserves (lipids and protein) similarly during diapause. Nevertheless, regulation of carbohydrate and glycogen reserves seems to vary between the populations: major peaks of these stored nutrients occur on different dates in the two populations, suggesting a differential regulation. Differences in diapause intensity imply a genetic differentiation between the two populations. The importance of our findings in understanding the physiological patterns during obligatory diapause of a univoltine insect species, as well as the practical implications for the development of specific phenological models for the European cherry fruit fly are discussed.

Social interactions regulate resource utilization in a Tephritidae fruit fly.

Previous studies have suggested that social interactions (e.g., the actions and reactions elicited by the interaction of co-specific individuals) induce individual fruit flies (Tephritidae) to ingest more food, especially protein-rich food. Changes in feeding behavior related to social interactions have been associated with reproduction (e.g., when different sexes are present), reproductive facilitation (e.g., when two females interact) and stress and aggression (e.g., flies of the same sex, or crowdedness). The present study investigated the effect of social interaction on the feeding, longevity and resource management of the Ethiopian fruit fly, Dacus ciliatus. Single flies and pairs of flies (of the same or different sexes) were confined to a small arena (the PUB system), in which we measured the amount of liquid food ingested daily by each fly. In addition, we sampled flies of different ages, extracted and quantified their lipid and protein contents, and related individual metabolic contents to the ingestion of a fructose and protein hydrolysate solution. Results showed that individual ingestion was significantly higher in flies maintained in pairs than in flies kept as solitary individuals. The highest intake rates were observed for the female-female pairs. In general, females ingested significantly greater volumes than males. Lipid contents tended to decrease progressively with age in flies kept as solitary individuals, especially in female flies, while lipid levels decreased and then increased in flies maintained in pairs. Protein trends were similar, although less pronounced than the patterns observed for the lipids. The flies kept as solitary individuals lived significantly longer than those kept in pairs. A resource-management analysis points to a decreased metabolic rate in flies kept as solitary individuals, as compared to paired flies. Results are discussed in view of theories of resource management and survival strategies.

Identification of host attractants for the ethiopian fruit fly, Dacus ciliatus loew.

The Ethiopian fruit fly, Dacus ciliatus, is an oligophagous pest of cucurbit crops, particularly melons, cucumbers, and marrows (summer squash). The present study aimed to identify host attractants for D. ciliatus and was guided by a behavioral bioassay and an electrophysiological assay. We tested volatile compounds from the fruits of a host plant, ripe and unripe Galia melon, Cucumis melo var. reticulates. Both sexes were attracted to melon volatiles. Those of ripe melon were preferred. Gas chromatography-electroantennographic detection analysis of the behaviorally active ripe melon volatiles consistently showed that 14 compounds elicited similar antennal responses from both sexes. Twelve compounds were identified by gas chromatography-mass spectrometry (GC-MS) using GC-MS libraries, retention indices (RI), and authentic standards. The electrophysiological activities of the compounds that were present at sufficient levels for identification, benzyl acetate, hexanyl acetate, (Z)-3-hexenyl acetate, (Z)-3-octenyl acetate, octanyl acetate, (Z)-3-decenyl acetate, and (E)-beta-farnesene, were evaluated at six different dosage levels by using electroantennography (EAG). Benzyl and hexanyl acetates elicited dose responses only in males, while other tested compounds elicited dose responses in both sexes. The strongest responses were observed for doses between 100 ng and 10 microg. The dose response, in terms of attractiveness to synthetic compounds within the active range (as determined by EAG), also was evaluated in the behavioral bioassay. Synthetic acetates were attractive to both sexes when tested individually. Significant attraction was observed when individual compounds were applied in the bioassay arena at doses of 0.5-1 microg/dispenser. Blends of compounds in equal proportions also were attractive to the insects. The most attractive blend was a mixture of four or five identified acetates. The addition of an equal proportion of (E)-beta-farnesene to this mixture had a deterrent effect.

Trends in lipid and protein contents during medfly aging: an harmonic path to death.

Survival and egg-laying trends were investigated in Mediterranean fruit flies (Ceratitis capitata) adults maintained on a sucrose-only diet, or on a full diet that consisted of a 3:1 sucrose and yeast hydrolizate mixture. In addition, we followed the total individual lipid and protein contents of aging flies in a cohort. Survival trends and life expectancy parameters at eclosion for males and females on full diet and for males on sucrose only were very similar. In contrast, the mortality of females on sucrose only was high early in life, but then slowed down. Egg-laying was ten times greater in female flies on full diet than in flies on sucrose only. Lipid contents in males and females on both types of diets were very similar, and harmonically oscillated with an amplitude of approximately 10 days. Successive crests of lipids tended to be smaller with the ageing of the cohort, and lipids contents significantly dropped at very advanced ages and close to the maximal age of the whole cohort. Protein contents of flies maintained on a full diet were high and stayed at a constant level throughout the entire life of the cohort. Protein levels in males and females on sucrose only dropped drastically during the first days of adult life, but then stayed stable at a low level until advanced ages. We propose that the synchronous rhythmic oscillation in lipid contents of male and female flies seems to be independently set by an internal clock. Protein reserves are allocated according to the access to protein food sources and these levels of protein are closely associated to egg production and mortality. Our results are discussed in view of resource allocation during reproduction and senescence.

Mediterranean fruit fly as a potential vector of bacterial pathogens.

The Mediterranean fruit fly (Ceratitis capitata) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit flies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female flies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit fly to serve as a vector for transmission of human pathogens to fruits. Addition of green fluorescent protein (GFP)-tagged Escherichia coli to a Mediterranean fruit fly feeding solution resulted in a dose-dependent increase in the fly's bacterial load. Flies exposed to fecal material enriched with GFP-tagged E. coli were similarly contaminated and were capable of transmitting E. coli to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the E. coli. Flies inoculated with E. coli harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of fluorescent bacteria were confined along the pseudotrachea in the labelum edge of the fly proboscis. Wild flies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identification of E. coli was made. These findings support the hypothesis that the common Mediterranean fruit fly is a potential vector of human pathogens to fruits.

Lipid and protein loads in pupating larvae and emerging adults as affected by the composition of Mediterranean fruit fly (Ceratitis capitata) meridic larval diets.

The effects of sucrose and amino acid (aa) composition and concentration in meridic larval diets (e.g., partially defined at the chemical level) was examined on several parameters of Mediterranean fruit fly (Medfly) development. Lipid and protein levels of pupating larvae and emerging adults were examined. Different sucrose concentrations in the diet had small effects upon most of the development parameters. However, sucrose concentration significantly affected the ability of larvae to accumulate lipid reserves and proteins. Adults emerging from the different sucrose diets did not significantly differ in their lipid contents and protein loads. Specific deletions of aa from the diet, and general aa concentration, had a strong effect upon the parameters of development and pupating larvae lipids and proteins. Glycine-deletion was the most deleterious, followed by the deletion of all non-essential aa, and serine. High aa concentration in the diet has a detrimental effect upon development. Lipid contents in pupating larvae, and to some extent protein levels, were affected by aa manipulations in the diet. Lipid and protein loads in emerging adults were not significantly affected by aa manipulations. Based on the analysis of lipid frequency distribution it is suggested that the Medfly seems to regulate the level of lipid content in emerging adults within a certain range, regardless of the larval diet history or lipid contents. Proteins do not seem to be regulated as are lipids. These results point to an interesting and unexpected metabolic regulation of energetic resources during metamorphosis of the Medfly.