Functional analysis of a fatty acid binding protein produced by Aphidius ervi teratocytes

Aphidius ervi (Hymenoptera, Braconidae) is an endophagous parasitoid of various aphid species, including Acyrthosiphon pisum (Homoptera, Aphididae), the model host used in the present study. Parasitized hosts show a marked increase of their nutritional suitability for the developing parasitoid larvae. This alteration of the biochemical and metabolic profile is due to a castration process mediated by the combined action of the venom, injected at the oviposition, and of the teratocytes, cells deriving from the dissociation of the embryonic membrane. Teratocytes produce and release in the host haemocoel two parasitism-specific proteins, which are of crucial importance for the development of their sister larvae. One of the proteins is a fatty acid binding protein (Ae-FABP), which shows a high affinity for C14-C18 saturated fatty acids (FAs) and for oleic and arachidonic acids. To better define the possible nutritional role of this protein, we have studied its immunolocalization profile in vivo and the impact on FA uptake by the epidermal and midgut epithelia of A. ervi larvae. During the exponential growth of A. ervi larvae, Ae-FABP is distributed around discrete lipid particles, which are abundantly present in the haemocoel of parasitized host aphids and in the midgut lumen of parasitoid larvae. Moreover, a strong immunodetection signal is evident on the surface of the two larval epithelia involved in nutrient absorption: the parasitoid midgut epithelium and the external epidermal layer. These two epithelia can effectively absorb radiolabelled myristic acid, but the FA transport rates are not affected by the presence in the medium of Ae-FABP. The protein appears to act essentially as a vector in the host haemolymph, transferring FAs from the digestion sites of host lipids to the growing parasitoid larvae. These data indicate that the proteins produced by A. ervi teratocytes may play complementary roles in the nutritional exploitation of the host.     

Host regulation by the aphid parasitoid Aphidius ervi: the role of teratocytes

The biochemical profile and metabolism of Acyrthosiphon pisum(Harris) (Homoptera, Aphididae) are markedly altered and redirected in response to parasitization by the endophagous braconid Aphidius erviHaliday (Hymenoptera, Braconidae). In the present study, the role played in the host regulation process by teratocytes, cells deriving from the dissociation of the embryonic membrane of the parasitoid, is taken into consideration. The protein synthesis activity of these cells of embryonic origin is analysed in vitroand an essential characterization of those proteins de novosynthesized and released in the incubation medium is provided. Teratocytes, obtained by dissecting parasitized host aphids, 3, 4 and 5 days after parasitoid oviposition, were incubated in vitroand, at the end of the incubation period, were separated from the medium for SDS-PAGE analysis of both cellular and secreted proteins. Various cellular proteins were more abundant as the time between parasitization and teratocyte collection increased. Furthermore, two proteins, showing an approximate molecular mass of 15 kD (p15) and 45 kD (p45) respectively, were abundantly secreted in the incubation medium by 5 day-old teratocytes. Incubations in presence of ³⁵S radiolabelled amino acids indicated that p15 and p45 are both synthesized by A. erviteratocytes. The amino acid composition of these two proteins was similar to that reported for other insect proteins with a demonstrated nutritional function. The p45 protein was found to be glycosylated. A tentative physiological model describing the host regulation role played by different parasitoid-derived factors is proposed.     

Nutrient absorption by Aphidius ervi larvae

It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) host regulation by the parasitoid larva induces in the aphid haemolymph major changes of the titer of nutritional compounds such as proteins, acylglycerols and free amino acids, in order to meet the stage-specific demands of the developing larva. Since little is known about how the larva absorbs these mobilized nutritional resources, nutrient absorption by larval stages of A. ervi was studied. In 2nd instar larvae, leucine was ten-fold accumulated in the haemocoel, and tyrosine and glutamine two-fold. Glucose and fructose were readily absorbed and fructose was extensively metabolized by larval tissues. In 3rd instars, the presence of a number of larvae that did not ingest the incubation medium enabled us to determine the respective amounts of substrate absorbed by the epidermis and the midgut. An accumulation of leucine in the haemocoel was observed only when midgut cells were involved in absorption, while the amino acid concentration within body fluids never exceeded that of the incubation medium when the uptake was performed only by epidermal cells. The immunofluorescence analysis, the mutual inhibition exerted on labeled glucose or fructose uptakes by a 100-fold excess of the sugars and the strong inhibition of uptakes induced by 0.2mM cytochalasin B support the expression of facilitative GLUT2-like transporters in the apical and basal cell membranes of midgut epithelial cells. Taken together, these results prove that both midgut and epidermis are involved in nutrient absorption throughout the parasitoid development, that GLUT2 transporters are responsible for glucose and fructose uptakes and that the chemical gradient that favors the passive influx of the two sugars is maintained by their conversion to other substrates.     

Host castration by Aphidius ervi venom proteins

The braconid Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera, Aphididae). Parasitized host aphids show different degrees of castration, a response that is total when parasitoid oviposition takes place in first instar hosts. Deleterious effects on the host reproductive system are already evident by 24h following parasitization, before egg hatching. The effect of parasitoid venom on A. pisum ovaries has been studied by performing microinjections in non-parasitized host aphids and observing the cellular alterations of the apical germaria of ovarioles. Venom injection reproduced the same alterations observed in parasitized aphids, while injections of saline solution did not induce any detectable change. By 24h, the germarial cells of both venom-treated aphids and parasitized aphids showed the absence of the nucleolus and of electron-dense material around the nucleus, frequently referred to as "nuage material". By 48h more evident signs of degeneration were observed, suggesting the possible occurrence of apoptosis. The bioactive component of the venom was both heat- and protease-sensitive. The activity was found in purified fractions that were highly enriched in two proteins with an approximate molecular mass of 21kD and 36kD, respectively. These macromolecules are the most abundant components of A. ervi venom and, unlike many venom proteins of studied parasitic Hymenoptera, are not glycosylated and appear to be subunits of an oligomeric protein. The adaptive significance of host castration is discussed.     

Fitness consequences of low temperature storage of Aphidius ervi

In biological control programs, storage at low temperatures is used to produce large number of individuals before inundative releases. However, an exposure to low temperatures may affect the survival of exposed individuals and some life history traits such as the daily fecundity or the longevity of the survivors. This impact on life history traits is important because they will determine the growth potential of the population. To estimate the costs of different treatments of low temperature exposure, we analyzed the main instantaneous demographic parameters of the parental generation of Aphidius ervi Haliday (Hymenoptera: Braconidae: Aphidiinae) when exposed to three storage temperatures (7, 4 and 0 °C), combined with two storage durations (one and two weeks) and two thermal regimes (constant and fluctuating), which represents a total of 12 treatments and a control. The average cohort generation time (Tc) decreased significantly after exposure to low temperature, particularly in fluctuating treatments. This means that development continues during storage. Cumulative fecundity and the net reproductive rate (R ₀ ) decreased significantly for most of the storage treatments. This decrease was most pronounced in the constant temperature treatments. Females under fluctuating thermal regime produced a higher cumulative fecundity than those exposed to constant temperatures. Surprisingly, an exposure to 0 °C over one week did not affect emergence rate and fecundity whereas all other treatments did. Storage at low temperatures may affect the efficiency of inundative releases, with the exception of storage for one week at 0 °C. Moreover, cumulative fecundity and net reproductive rate (R ₀ ) represent a more immediate prediction of the increase of the population after low temperature exposure than the intrinsic rate of increase (r _m ), and reflect the qualities required in an inundative release context.     

Responses to aphid sex pheromones by the pea aphid parasitoids Aphidius ervi and Aphidius eadyi

Aphidius ervi and Aphidius eadyi, two parasitoids of the pea aphid Acyrthosiphon pisum, were attracted to components of the aphid sex pheromone in laboratory bioassays. Pre-test experience with host aphids in the presence of aphid sex pheromone did not affect the response of A. ervi to pheromone in a 4-way olfactometer, compared with that of naive parasitoids. Aphidius ervi females exposed only to the pheromone prior to testing did not respond in the olfactometer, suggesting habituation to the foraging cue by the parasitoid. In a wind tunnel, aphid sex pheromone increased the attraction of A. ervi to the plant-host complex (Vicia faba/A. pisum), suggesting an additive effect when two different foraging cues are present simultaneously.     

Pea aphid clonal resistance to the endophagous parasitoid Aphidius ervi

The physiological mechanism of resistance to the endophagous braconid Aphidius ervi Haliday (Hymenoptera, Braconidae) by a pink clone (PC) of Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) has been investigated. Comparative data on parasitoid development and associated host biochemical changes in the resistant PC aphids and in a susceptible green clone (GC) of A. pisum are reported. When the PC aphids were attacked as early 4th instars, the developing parasitoid larvae showed a strongly reduced increase in size, compared to those synchronously developing in GC aphids, and were unable to produce a regular mummy. In contrast, parasitism of 2nd instar PC aphids, allowed completion of parasitoid development, but adults had a prolonged developmental time, due to a longer duration of parasitoid’s final (3rd) instar. In all cases, teratocytes, cells deriving from the A. ervi serosal membrane, and the proteins abundantly synthesised by them, were never found in the haemolymph of parasitised PC aphids. Host castration, as demonstrated by total protein incorporation into reproductive tissues, was total in the majority of early (2nd instar) parasitised host aphids, while it was limited when later instars (4th) of PC aphids were parasitised. This is partly due to the absence of the cytolytic activity of teratocytes on host embryos, which, through their persistence, may compete for nutritional resources with the developing parasitoid larvae. In parasitised PC aphids, this competitive effect is further aggravated for the parasitoid by the absence of the regulated amino acid titre increase in the host haemolymph, which is regularly observed in GC aphids. Failure of teratocyte development in the PC clone of the pea aphid is, then, the major functional constraint accounting for the reduction/inhibition of A. ervi larval growth. The reported results allow to assess in vivo the role of teratocytes in the host physiological redirection and nutritional exploitation by the parasitoid, and to integrate and validate the proposed physiological model of host-parasitoid interactions in the system A. pisum-A.ervi.     

An 'artificial aphid' for Aphidius ervi (Hym., Braconidae)

Abstract:  An artificial aphid was developed by making parafilm^® bubbles filled with aphid haemolymph and its suitability for parasitoid oviposition was tested. Aphidius ervi accepted the parafilm^® aphid and laid eggs on it. By stretching, the thickness of the parafilm could be changed. The thicker the parafilm, the higher was the mean number of ovipositional stings and the lower the mean number of eggs released by A. ervi .     

Reproductive strategies of Aphidius ervi Haliday (Hymenoptera: Aphidiidae)

Hymenopteran parasitoids are usually arrhenotokous parthenogenetic, where females arise from fertilized and males from unfertilized eggs. Therefore, the reproductive fitness of females is a function of egg production and furthermore affected by mating, whereas that of males is mainly determined by the number of daughters they father. Aphidius ervi Haliday is a quasi-gregarious parasitoid of a number of aphid pests on economically important crops such as legumes and cereals. Females are monandrous whereas males are polygynous. Here, we tested how parental age at mating and male mating history affected mating success, fecundity and daughter production in this species. Once-mated males perform significantly better than naïve males with regard to mating success, suggesting that males learn from previous matings. The fecundity of virgin females is not significantly different from that of mated females regardless of parental age at mating and male mating history, indicating that mating does not stimulate egg production or contribute to female nutrient supply. Males can replenish sperm supply after mating, implying that they are at least moderately synspermatogenic. Preference for young over old mates for mating by both sexes may be explained by the fact that aging of both sexes contributes to the reduction of daughter production. Rather than sperm depletion, the reduced daughter production may be attributed to diminishing sperm viability and mobility in aging males and increasing constraints in fertilization process in aging females. Our results also show that female age has a stronger impact on the production of daughters, suggesting that fertilization process in females is more sensitive to aging than sperm vigor in males.     

Functional bases of host-acceptance behaviour in the aphid parasitoid Aphidius ervi

Abstract.  The host acceptance behaviour in Aphidius ervi is investigated, assessing the role of both external and internal host-associated cues, offered to the experimental parasitoids with parafilm-made aphid dummies. The reaction to internal cues present in the host haemolymph is clearly evident, and its intensity is enhanced by external cues. Parasitoid females lay few eggs in aphid dummies filled with host haemolymph. A significant increase in the number of both oviposition reactions (host stinging) and egg laying is observed only when these dummies are coated with cornicle secretion. However, this enhancement is not observed when the aphid dummies contain distilled water. Thus, the host acceptance behaviour of A. ervi females appears to be controlled by the integration of both external and internal chemical cues. The physiological basis of this behavioural response is investigated with a detailed study on the anatomy and ultrastructure of A. ervi ovipositor. The detection of chemical cues present in the host haemolymph that act as kairomones is made possible by the presence of gustatory sensilla on the tip of the ovipositor. These sensilla consist of porous areas, reached by unbranched dendrites running inside both the lower valves (i.e. first valvulae) and the unpaired upper valve (i.e. second valvulae). The mechanosensory function during oviposition appears to be provided mostly by the basiconic sensilla found on the tip of external valves (i.e. third valvulae). A tentative functional model accounting for the observed oviposition behaviour of A. ervi is proposed.     

Physical and chemical cues influencing the oviposition behaviour of Aphidius ervi

The oviposition behaviour of the aphid parasitoid Aphidius ervi Haliday is influenced by both chemical and physical cues. Oviposition attack responses were elicited by paint pigments sealed into the tip of a glass capillary tube. Parasitoids reacted to yellow pigments with repeated oviposition attack responses, but they did not react to green pigments. The spectrum of reflected light from the yellow pigments was very similar to that from the `green' natural host Acyrthosiphon pisum (Harris), with a high proportion of the total radiation energy being emitted in the yellow-orange wavebands (580–660 nm). Pea aphid cornicle secretion also elicited oviposition attack responses, which were not exclusively induced by its pale yellow-green colour. In fact, the oviposition attack response to capillary tips coated with cornicle secretion remained evident under red light conditions, which, in contrast, nearly completely suppressed the response to yellow pigments. Chemical compounds from cornicle secretion do not appear to be involved in parasitoid orientation, even though they stimulate intense oviposition attack responses. Olfactometer experiments showed that the putative kairomone involved acts only at very short range or on contact. Host exuviae, which also elicited strong and persistent oviposition reactions from A. ervi females, appear to be a good alternative source of ovipositional kairomone(s). This work confirms the existence of an aphid cuticular kairomone.     

Decision to superparasitize based on larval survival: competition between aphid parasitoids Aphidius ervi and Aphidius smithi

Oviposition by a parasitoid wasp in an already parasitized host (= superparasitism) may result in larval competition and the loss of offspring. Consequently, in solitary species, the decision to superparasitize should be based on the probability of offspring survival. Females of Aphidius ervi Haliday and A. smithi Sharma & Subba Rao (Hymenoptera: Aphidiidae), two parasitoids of the pea aphid, discriminated between conspecific-and heterospecific-parasitized hosts. Both species showed partial preference for aphids previously attacked by A. smithi, a result predicted from the fact that A. ervi is superior to A. smithi in larval competition under most conditions. Females of A. smithi selectively attacked conspecific-parasitized pea aphids when given a choice between these and self-parasitized hosts. Results indicated that females of both species responded to an external pheromone-like marker to discriminate between selfconspecific-and heterospecific-parasitized aphids. In addition, A. smithi may use internal cues to recognize, and avoid oviposition in, aphids previously parasitized by A. ervi.

---

Résumé L'oviposition par une guêpe parasite dans un hôte qui a déjà été parasité (= superparasitisme) pourrait conduire à une compétition entre larves et à une perte de progéniture. Par conséquent, dans les espèces solitaires, la décision de superparasiter devrait être basée sur la probabilité de survie de la progéniture. Les femelles de Aphidius ervi Haliday et de A. smithi Sharma & Subba Rao (Hymenoptera: Aphidiidae), deux parasites du puceron du pois, ont discriminé entre les hôtes parasités par des individus de la même espèce et ceux parasités par des individus d'une espèce différente. Les deux espèces de parasite ont montré une préférence partielle pour les pucerons préalablement parasités par A. smithi, résultat qui était prévisible du fait que, dans la majorité des cas, A. ervi est supérieur à A. smithi dans la compétition entre larves. Les femelles de A. smithi ont sélectivement attaqué les pucerons parasités par un individu de la même espèce lorsqu'il leur était donné de choisir parmi ceux-ci et ceux parasités par les même individu. Les résultats indiquaient que les femelles des deux espèces répondaient à une sorte de phéromone externe pour distinguer les pucerons parasités par le même individu de ceux parasités par un individu de la même espèce et ceux parasités d'une autre espèce. En plus, A. smithi pourrait utiliser des signaux internes pour reconnaître et éviter l'oviposition dans les pucerons préalablement parasités par A. ervi.

     

Effects of honeydew sugar composition on the longevity of Aphidius ervi

Feeding on sugar‐rich foods such as nectar and honeydew is important for survival of many adult parasitoids. Especially in agricultural systems, honeydew is often the most prevalent carbohydrate source. However, relative to plant nectar, honeydew may be relatively unsuitable, as a result of an unfavourable sugar composition or the presence of secondary plant compounds. We studied survival of the aphid parasitoid Aphidius ervi Haliday (Hymenoptera: Braconidae) on honeydew collected from various aphid species feeding on potato (Solanum tuberosum L., cv. Desiree) (Solanaceae), wheat (Triticum aestivum L., cv. Bobwhite) (Poaceae), or artificial diet, as well as the sugar composition of the different honeydews. Honeydews from the tested aphid species on potato, wheat, or artificial diet were found to be relatively suitable food sources for adult A. ervi, although not always as suitable as a 2 M sucrose solution. There were differences in honeydew sugar composition among the different aphid species on the various host plants. Multivariate statistics showed that the factor ‘aphid species’ had a significant influence on the sugar composition of the honeydew, explaining 27% of the variation in the potato system and 89% in the wheat system. When exploring the relationship between carbohydrate composition of the honeydews from aphids on potato and wheat plants, and their nutritional value for A. ervi, data revealed that differences in parasitoid longevity can to some extent be explained by carbohydrate composition. Furthermore, our results confirm that sucrose and its hexose components glucose and fructose are very suitable carbohydrate sources for hymenopteran parasitoids and show that parasitoid survival on an equimolar solution of the two monosaccharides glucose and fructose does not exceed performance on the disaccharide sucrose.     

Host-locating response by the aphid parasitoid Aphidius ervi to tomato plant volatiles

Abstract

The blend of volatile compounds emitted by tomato plants (Solanum lycopersicum) infested with the potato aphid (Macrosiphum euphorbiae) has been studied comparatively with undamaged plants and aphids themselves. Aphid-infested plants were significantly more attractive towards Aphidius ervi than undamaged plants and aphids themselves. Oriented response towards host-damaged plant, from which aphids were removed just before running the bioassay, did not differ from that recorded for infested plants. Collection of the volatiles and analysis by gas chromatography revealed only quantitative differences between uninfested and aphid-infested plants. Nine compounds, α-pinene, (Z)-3-hexen-1-ol, α-phellandrene, limonene, (E)-β-ocimene, p-cymene, methyl salicylate, (E)-β-caryophyllene and an unknown compound, were emitted at higher levels from aphid-infested plants than from undamaged control plants, whilst no differences were noted for hexanal, 6-methyl-5-hepten-2-one, and humulene (=α-caryophyllene). Synthetic standards of these compounds were tested in wind tunnel bioassays and all elicited a significant increase in oriented flight and landings on the target by the aphid parasitoid Aphidius ervi. (E)-β-caryophyllene resulted the most attractive towards female wasps. These results corroborate the hypothesis that the volatiles produced by the plant in response to aphid attack derive from both jasmonic and salicylic acid pathways, and are exploited by A. ervi as olfactory cues to locate its hosts.     

A comparison between two strains from Japan and Europe of Aphidius ervi

A European strain of Aphidius ervishall be introduced into Japan for the control of Macrosiphum euphorbiaeon greenhouse tomatoes. We compared this strain, which is originating from The Netherlands (EVP), with a native strain from Hokkaido, Northern Japan (SPR) in several characteristics. The percentage of parasitism of SPR and EVP was significantly different, when attacking three aphid species: 13% and 30% on M. euphorbiaeon tomato, 68% and 93% on Acyrthosiphon pisumon broad bean and 10% and 42% on Aulacorthum solanion sweet pepper, respectively. SPR and EVP differed morphologically (in the colouration of the petiole) and in the esterase banding patterns. Furthermore, cross mating experiments showed that SPR and EVP are partially reproductively isolated from each other. Complete reproductive isolation is evident in the one direction cross (EVP females and SPR males).     

Absorption of sugars and amino acids by the epidermis of Aphidius ervi larvae

Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of several aphid species of economic importance, widely used in biological control. The definition of a suitable artificial diet for in vitro mass production of this parasitoid is still an unresolved issue that, to be properly addressed, requires a deeper understanding both of its nutritional needs and of the functional properties of the larval epithelia involved in nutrient absorption. The experimental evidence presented in this paper unequivocally demonstrates that the uptake of sugars and amino acids takes place through the body surface of the larval stages of A. ervi. These nutrients are efficiently absorbed by the larval epidermis, but the transport rate progressively declines over time. The epidermis exhibits a cross-reactivity to antibodies raised against the mammalian facilitative glucose transporter GLUT2 and the sodium cotransporter SGLT1. The analysis of sugar transport sensitivity to specific inhibitors indicates the involvement of GLUT2-like transporters, while a role for SGLT1-like transporters is not supported. The peculiar pathways of nutrient absorption in A. ervi larvae further corroborate the general idea that the pre-imaginal stages of endophagous koinobiont Hymenoptera, like Metazoan parasites, show a high degree of physiological integration with their hosts.     

Behavioural differences between Aphidius ervi populations from two tritrophic systems are due to phenotypic plasticity

The Palaeoarctic parasitoid Aphidius ervi Haliday (Hymenoptera, Aphidiidae) parasitises legume aphids in its region of origin. In Chile, it parasitises both legume and cereal aphids. This special situation was studied at two levels: (i) the host searching behaviour of A. ervi from two different tritrophic systems (Acyrthosiphon pisum on alfalfa and Sitobion avenae on wheat) was investigated in dual choice tests in a wind tunnel between odours from both A. pisum-alfalfa host plant complex (HPC) and S. avenae-wheat HPC, and (ii) the genetic structure of A. ervi populations from both sources using molecular markers. Responses of A. ervi females to volatile olfactory cues emanating from A. pisum-alfalfa HPC and S. avenae-wheat HPC were significantly higher towards the HPC on which they were reared during the last generation before experimentation, regardless of the origin of the parasitoid. As previously described for this parasitoid species, oviposition experience was also of major relevance in the preferences of female parasitoids. On the other hand, variation in mitochondrial DNA segments and RAPD-PCR polymorphism using total DNA showed the absence of host-based population structure and a high genetic homogeneity between these A. ervi populations. These results reject the possible existence of different host-strains of this parasitoid in Chile.     

Olfactory conditioning in mate searching by the parasitoid Aphidius ervi (Hymenoptera: Braconidae)

Despite the fact that insect learning capacity has been broadly demonstrated, the role that this process plays during mate searching has been scarcely explored. We studied whether the sexual behaviour of a male parasitic wasp can be conditioned to the odours from two alternative host plant complexes (HPCs) present during its first copulation. The experimental subjects were newly emerged males of the aphid parasitoid, Aphidius ervi, and two alternative HPCs (alfalfa or wheat). In the training protocol, copulation experience corresponded to an unconditioning stimulus and HPC odours to the conditioning stimuli. The initial (just after eclosion) and trained responses were assessed in a glass Y-olfactometer. The results showed that neither alfalfa HPC nor wheat HPC stimuli elicited sexual-related behaviours in initial male responses. Conversely, both HPCs triggered strong attraction and wing fanning courtship behaviour in trained responses when the male was exposed to a female plus HPC during training. In males trained with females plus a given HPC but tested with the alternative HPC in the olfactometer, trained response showed a similar trend to the non-associative treatments. Hence, through learning, the olfactory stimulus context present during copulation could become a predictive cue for further mate searching. These results are discussed in terms of parasitic wasp ecology and host fidelity.