Mapping candidate genes for Drosophila melanogaster resistance to the parasitoid wasp Leptopilina boulardi

Drosophila melanogaster resistance against the parasitoid wasp Leptopilina boulardi is under the control of a single gene (Rlb), with two alleles, the resistant one being dominant. Using strains bearing deletions, we previously demonstrated that the 55E2-E6; 55F3 region on chromosome 2R is involved in the resistance phenomenon. In this paper, we first restricted the Rlb containing region by mapping at the molecular level the breakpoints of the Df(2R)Pc66, Df(2R)P34 and Df(2R)Pc4 deficiencies, using both chromosomal in situ hybridization and Southern analyses. The resistance gene was localized in a 100 kb fragment, predicted to contain about 10 different genes. Male recombination genetic experiments were then performed, leading to identification of two possible candidates for the Rlb gene. Potential involvement of one of this genes, edl/mae, is discussed.     

Reduced cellular immune competence of a temperature-sensitive dopa decarboxylase mutant strain of Drosophila melanogaster against the parasite Leptopilina boulardi.

1. The melanotic encapsulation response made by larvae of a temperature-sensitive dopa decarboxylase (DDC) mutant strain of Drosophila against the parasitic wasp Leptopilina was severely compromised in hosts with reduced levels of DDC. 2. Dopa and 5,6-dihydroxyindole (DHI) were two hemolymph components identified in hosts exhibiting a melanotic encapsulation response. 3. This is the first study to implicate DDC in insect cellular immune responses, and to provide chemical evidence that the pigment formed during such responses is eumelanin derived from tyrosine.     

Two non-linked genes for specific virulence of Leptopilina boulardi against Drosophila melanogaster and D. yakuba

The developmental success of Leptopilina boulardi parasitoids within host species of the melanogaster subgroup is determined mainly by their ability to suppress the host immune reaction (virulence). Host resistance and parasitoid virulence are genetically variable in both partners. A gene for specific resistance against L. boulardi (Rlb) has been identified in Drosophila melanogaster, and a gene for the immune suppression (IS) of D. melanogaster has been identified in L. boulardi. To understand the evolution of the IS gene, we determined its specificity regarding potential hosts of the melanogaster subgroup. It did not affect the virulence against any other species of the melanogaster subgroup and was called ISm for immune suppression of D. melanogaster. Another gene (ISy), non-linked to the gene ISm, was characterized for the specific immune suppression of D. yakuba. These results suggesting that natural selection for virulence against one host species does not influence the evolution of virulence against another will allow us to develop pertinent hypotheses concerning the evolution of this character which is expected to drive the evolution of the parasitoid toward narrow host specialization. This revised version was published online in July 2006 with corrections to the Cover Date.     

Active suppression of D. melanogaster immune response by long gland products of the parasitic wasp Leptopilina boulardi

To develop inside their insect hosts, endoparasitoid wasps must either evade or overcome the host's immune system. Several ichneumonid and braconid wasps inject polydnaviruses that display well-studied immune suppressive effects. However, little is known about the strategies of immunoevasion used by other parasitoid families, such as figitid wasps. The present study provides experimental evidence, based on superparasitism and injection experiments, that the figitid species Leptopilina boulardi uses an active mechanism to suppress the Drosophila melanogaster host immune response, i.e. the encapsulation of the parasitoid eggs. The immune suppressive factors are localised in the long gland and reservoir of the female genital tractus, where virus-like particles (VLPs) have been observed. Parasitism experiments using a host tumorous strain indicate that these factors do not destroy host lamellocytes but that they impair the melanisation pathway. Interestingly, they are not susceptible to heating and are not depleted with prolonged oviposition experience, in contrast to observations reported for L. heterotoma, another figitid species. The mechanisms that prevent encapsulation of eggs from L. boulardi and L. heterotoma differ in several respects, suggesting that different physiological strategies of immunosuppression might be used by specialised and generalist parasitoids.     

Resistance of Drosophila suzukii to the larval parasitoids Leptopilina heterotoma and Asobara japonica is related to haemocyte load

Unlike other Drosophila species, the invasive Drosophila suzukii Matsumura (Diptera: Drosophilidae) shows a remarkable pest status. Among the physiological traits that may explain the high level of resistance to parasitoids of Drosophila larvae, the haemocyte load is shown repeatedly to play an important role. To determine whether haemocyte load can explain immunity resistance of D. suzukii to parasitoids, the haemocytes of parasitized and healthy larvae are quantified in two Japanese and three French populations of D. suzukii. Parasitization tests are conducted with two larval parasitoids: the paleartic Leptopilina heterotoma Thomson (Hymenoptera: Figitidae) and the Asian Asobara japonica Belokobylskij (Hymenoptera: Braconidae). Based on morphological and functional criteria, D. suzukii has classes of haemocytes similar to those described in Drosophila melanogaster. However, healthy larvae of the five populations tested possess particularly large numbers of haemocytes compared with D. melanogaster. Haemocyte load is also higher in larvae from the French populations than in the Japanese strains. The ability of D. suzukii larvae to encapsulate eggs of L. heterotoma is associated with a particularly high load of circulating haemocytes. However, it is notable that A. japonica induces a strong depression of the haemocyte population in this resistant host associated with an inability to encapsulate parasitoid eggs. The results show that the cellular immune system plays a major role in the failure of larval parasitoids to develop in most instances in larvae of D. suzukii, possibly contributing to the success of this species as an invader.     

Structure and electrophysiological responses of gustatory organs on the ovipositor of the parasitoid Leptopilina heterotoma

Location, structure and histology of chemosensilla on the tip of the ovipositor of the parasitoid Leptopilina heterotoma are described based on SEM and TEM studies. Furthermore, we developed a method for recording extracellular action potentials from the gustatory neurons in response to host haemolymph. This method allowed us to record multi-unit recordings from a sensillum occurring singly on the unpaired ovipositor valve. The TEM study of the ovipositor tip revealed the presence of six dendrites, the electrophysiological recordings provided evidence for the activity of three or possibly four gustatory neurons in response to the complex stimulus offered, leaving other taste functions or a mechanoreceptor function open for the remaining neurons.     

Haemocyte changes in resistant and susceptible strains of D. melanogaster caused by virulent and avirulent strains of the parasitic wasp Leptopilina boulardi

Two strains of Drosophila melanogaster (resistant and susceptible) were parasitized by a virulent or avirulent strain of the parasitoid wasp Leptopilina boulardi. The success of encapsulation depends on both the genetic status of the host strain and the genetic status of the parasitoid strain: the immune cellular reaction (capsule) is observed only with the resistant strain-avirulent strain combination. The total numbers of host haemocytes increased in all 4 combinations, suggesting that an immune reaction was triggered in all hosts. Resistant host larvae infected with the virulent or avirulent strains of parasitoid wasp had slightly more haemocytes per mm(3) than did susceptible host larvae at the beginning of the reaction (less than 15 h post-parasitization). This difference disappeared later. Only the virulent parasitoid strain caused the production of a high percentage of altered lamellocytes (from a discoid shape to a bipolar shape), half the total number of lamellocytes are altered. This suggests that the alteration of lamellocyte shape alone is not sufficient to explain the lack of capsule formation seen in resistant hosts parasitized by the virulent strain. Lastly, there were very few altered lamellocytes in resistant or susceptible hosts parasitized by the avirulent parasitoid strain, two combinations in which no capsule was formed. As is now established for Drosophila-parasitoid interactions, virus-like particles contained in the long gland of the female wasp affect the morphology of the lamellocytes. The results presented here are further proof of the action (direct or indirect) of virus like particles of the virulent strain on lamellocytes.     

Local adaptations of life‐history traits of a Drosophila parasitoid,Leptopilina boulardi: does climate drive evolution?

1. Climate is an important source of selection on life histories, and local adaptations to climate have been described in several cline studies. Temperature is the main climatic factor that has been considered as an agent of selection, whereas other factors may vary with it, such as precipitation. 2. We compared life‐history traits of five populations of Leptopilina boulardi, a Drosophila parasitoid, originating from contrasting climates. Referring to cline studies, we hypothesised shorter lifespan, earlier reproduction, and lower lipid content in populations from the hottest and driest areas if life histories have been selected in response to temperature and/or humidity. 3. Our results are opposite to these predictions. Females from humid and mild climates invested more in early reproduction and lived for fewer days than females from dry and hot areas, which were synovigenic (i.e. they matured additional eggs during adult life) and able to synthesise lipids during adult life. 4. We suggest that life histories are more adapted to host distribution than to climatic factors. Drosophila patches are more abundant in the humid area, allowing the parasitoids to spend less energy and time finding hosts. This may result in selection for early reproduction traded‐off against longevity. In the hot and dry climate, females have to fly large distances to find host patches. Synovigeny, a long lifespan, lipogenesis, and high dispersal ability may be adaptive there. This is the first time that between‐population differences in the ability to synthesise lipids have been described in parasitoids.     

Correlated responses to selection on body size in Drosophila melanogaster.

Correlated responses to artificial selection on body size in Drosophila melanogaster were investigated, to determine how the changes in size were produced during development. Selection for increased thorax length was associated with an increase in larval development time, an extended growth period, no change in growth rate, and an increased critical larval weight for pupariation. Selection for reduced thorax length was associated with reduced growth rate, no change in duration of larval development and a reduced critical larval weight for pupariation. In both lines selected for thorax length and lines selected for wing area, total body size changed in the same direction as the artificially selected trait. In large selection lines of both types, the increase in size was achieved almost entirely by an increase in cell number, while in the small lines the decrease in size was achieved predominantly by reduced cell size, and also by a reduction in cell number. The implications of the results for evolutionary-genetic change in body size in nature are discussed.     

Adaptive self- and conspecific superparasitism in the solitary parasitoid Leptopilina heterotoma (Hymenoptera: Eucoilidae)

One of the foraging decisions facing parasitoids is whetherto accept (superparasitize) or reject hosts that have alreadybeen parasitized. An important distinction is whether the hosthas been parasitized by the female parasitoid herself or bya conspecific. In solitary parasitoids, the pay-off from anegg laid in the latter host type (conspecific superparasitism)is the probability that the second egg wins the competitionfor the host and results in an offspring. The pay-off from anegg laid in the former type (self-superparasitism) increaseswith an increasing probability that another female will superparasitizethe host in the near future. When this probability equals one,self-superparasitism and conspecific superparasitism have thesame payoff. However, conspecific superparasitism will generallyhave a higher pay-off than self-superparasitism. It will thereforebe beneficial for a female parasitoid to be able to distinguishbetween a host she parasitized and one parasitized by a conspecific.The degree of benefit depends on the probability of conspecificsuperparasitism in the near future. Using an optimal diet model,I show that when a parasitoid encounters a patch containinga mixture of unparasitized and already-parasitized hosts, afemale that can distinguish between the two types of parasitizedhosts gains more offspring than a female without this ability.However, when parasitoids search a patch together with conspecifics,it is adaptive to self-superparasitize, and the pay-off fromthis ability may be negligible. It is therefore predicted thatwhen a female parasitoid searches a partially depleted patchalone, it will reject the hosts parasitized by itself more frequentlythan hosts parasitized by conspecifics. In contrast, femaleparasitoids searching together are predicted to accept hoststhat they parasitized themselves much more often. The resultsshow that the solitary parasitoid Leptopilina heterotoma (Hymenoptera:Eucoilidae) is able to distinguish between hosts that it parasitizedand hosts parasitized by conspecifics. The predictions of themodel are met in a second experiment that shows that L. heterotomaself-superparasitizes when the probability of conspecific superparasitismis high.     

Egg-laying experience and acceptance of parasitized hosts by the parasitoid,Leptopilina heterotoma (Hymenoptera: Eucoilidae)

The influence of egg-laying experience on the response of females of the eucoilid parasitoid,Leptopilina heterotoma, to parasitized and unparasitizedDrosophila melanogaster host larvae was examined under more controlled conditions than those used in past studies. In laboratory assays, we precisely manipulated both the number of eggs laid by females and the kind of larvae (parasitized versus unparasitized) in which the eggs were laid. We found that the tendency to avoid laying eggs in parasitized hosts depended markedly on whether or not eggs had been laid previously, but depended little on whether those eggs had been laid in parasitized or unparasitized hosts. The observed effect of general egg-laying experience on avoidance of parasitized hosts may reflect responses to either changes in the wasp's internal state (perhaps, changes in egg load) or changes in the wasp's neural representation of the external environment (such as those presumed to occur during learning). In light of these results, we offer a tentative reinterpretation of several earlier studies.     

Strong responses of Drosophila melanogaster microbiota to developmental temperature

Physiological responses to changes in environmental conditions such as temperature may partly arise from the resident microbial community that integrates a wide range of bio-physiological aspects of the host. In the present study, we assessed the effect of developmental temperature on the thermal tolerance and microbial community of Drosophila melanogaster. We also developed a bacterial transplantation protocol in order to examine the possibility of reshaping the host bacterial composition and assessed its influence on the thermotolerance phenotype. We found that the temperature during development affected thermal tolerance and the microbial composition of male D. melanogaster. Flies that developed at low temperature (13°C) were the most cold resistant and showed the highest abundance of Wolbachia, while flies that developed at high temperature (31°C) were the most heat tolerant and had the highest abundance of Acetobacter. In addition, feeding newly eclosed flies with bacterial suspensions from intestines of flies developed at low temperatures changed the heat tolerance of recipient flies. However, we were not able to link this directly to a change in the host bacterial composition.     

The attractiveness of different odour sources from the fruit–host complex on Leptopilina boulardi, a larval parasitoid of frugivorous Drosophila spp.

In parasitoid insects, successful offspring development depends on the female’s ability to find a suitable host. Specific recognition is often based on responses to olfactory cues, but their source and nature have rarely been determined. –This paper deals with the recognition of odours involved in host location by Leptopilina boulardi[Barbotin, Carton & Kelner-Pillault] (Hymenoptera: Eucoilidae), a larval parasitoid of Drosophila species that develops in mature fruits. The nature and origin of volatile stimuli recognized among odours of the host–fruit complex, and the effect of learning on this recognition, were investigated. Oriented responses to these odours were observed in a four-armed olfactometer and were analysed with the observer software (Noldus Information Technology). Fruit odours alone (banana and pear) were not spontaneously attractive to naive parasitoids, whereas naturally-infested bananas were highly attractive. The attraction was related to the odour that adult Drosophila left on the substrate but not to Drosophila oviposition activity or larval development. A synergism between some fruit odours (banana and pear) and the odour left by adult Drosophila on damp filter paper was observed. However, when testing a non-fruit substrate (mushroom), no synergism was observed. Thus, female L. boulardi may innately recognize host–food substrate odours associated with odours from the adult stage of their host. In addition, an oviposition experience on an infested banana allows L. boulardi females to memorise the fruit odour itself through associative learning. The adaptive significance of this process is discussed.     

LUSH odorant-binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster.

The molecular mechanisms mediating chemosensory discrimination in insects are unknown. Using the enhancer trapping approach, we identified a new Drosophila mutant, lush, with odorant-specific defects in olfactory behavior. lush mutant flies are abnormally attracted to high concentrations of ethanol, propanol, and butanol but have normal chemosensory responses to other odorants. We show that wild-type flies have an active olfactory avoidance mechanism to prevent attraction to concentrated alcohol, and this response is defective in lush mutants. This suggests that the defective olfactory behavior associated with the lush mutation may result from a specific defect in chemoavoidance. lush mutants have a 3-kb deletion that produces a null allele of a new member of the invertebrate odorant-binding protein family, LUSH. LUSH is normally expressed exclusively in a subset of trichoid chemosensory sensilla located on the ventral-lateral surface of the third antennal segment. LUSH is secreted from nonneuronal support cells into the sensillum lymph that bathes the olfactory neurons within these sensilla. Reintroduction of a cloned wild-type copy of lush into the mutant background completely restores wild-type olfactory behavior, demonstrating that this odorant-binding protein is required in a subset of sensilla for normal chemosensory behavior to a subset of odorants. These findings provide direct evidence that odorant-binding proteins are required for normal chemosensory behavior in Drosophila and may partially determine the chemical specificity of olfactory neurons in vivo.     

The joint effect of temperature and host species induce complex genotype-by-environment interactions in the larval parasitoid of Drosophila, Leptopilina heterotoma (Hymenoptera: Figitidae)

Phenotypic plasticity and genotype-by-environment interactions are usually studied by testing environmental factors separately. However, several environmental factors may vary simultaneously across the geographic range of a species, and thus interact to drive phenotypic and genotypic evolution. Here, we address the question of the interaction between two environmental factors in the case of a koinobiont parasitoid of Drosophila , Leptopilina heterotoma (Hymenoptera: Figitidae). Using two allopatric parasitoid lines, we analysed the joint effect of developmental temperature and host species on two major fitness components (pre-imaginal survival and fecundity) of emerged parasitoids. Our results indicate that both environmental factors strongly interact to shape the parasitoid phenotype. Moreover, complex genotype-by-environment interactions appeared that can even invert the relative fitness of the two parasitoid lines.     

Enzymatic responses of Drosophila melanogaster to long- and short-term exposures to ethanol

The effects of environmental ethanol on larva-to-pupa survival and on the activities of four enzymes were investigated in three Drosophila melanogaster strains. The strains had different allelic combinations at the Odh and Aldox loci on their third chromosomes, but they all carried the Adh ^S -Gpdh ^F allelic combination on the second chromosome. Replicates of each of the strains were exposed to three different ethanol treatments: (i) no ethanol in the medium (control); (ii) 5% ethanol for a single generation (short-term exposure); (iii) 5% ethanol for 20 generations (long-term exposure). In all experiments, the activities of four enzymes (ADH, ODH, GPDH and AOX) were measured in larvae, pupae and adults. The results showed that (i) the larval and adult metabolic responses to environmental ethanol were different; (ii) enzyme activity changes under short-term exposure differed from those measured under long-term exposure; (iii) the activities of the allozymes common to all strains (ADH-S and GPDH-F), differed depending on the genetic background. Changes in larva-to-pupa survival were seen when the larvae of control and exposed lines of the three strains were confronted with various concentrations of ethanol. In all three strains, the exposed lines had significantly higher initial survival rate and ethanol tolerance than the control lines. Strain-specific differences were observed in the ethanol tolerance of both types of line.