Maria Luisa Dindo

Drosophila parasitoids in northern Italy and their potential to attack the exotic pest Drosophila suzukii

Drosophila suzukii is an invasive alien pest recently introduced into Europe and North and South America. Several control methods have been tested, and the ability of natural enemies to control this pest has been investigated. This study aimed to identify the main parasitoids of drosophilids in North Italy via field surveys, and to evaluate the ability of some of those species emerged to parasitize D. suzukii compared to indigenous D. melanogaster. A nine-site survey from July to October 2014 that exposed fruit (banana and blueberry) for 7 and 14xa0days obtained six parasitoid species, ranked from highest abundance: Leptopilina boulardi, L. heterotoma (Hymenoptera: Figitidae), Pachycrepoideus vindemiae (Hymenoptera: Pteromalidae), Trichopria cf. drosophilae (Hymenoptera: Diapriidae), Asobara tabida (Hymenoptera: Braconidae), and Spalangia erythromera (Hymenoptera: Pteromalidae). The presence and abundance of these species varied greatly among the sites and across the season. The field survey results showed a relationship between parasitoids and indigenous Drosophila communities and a high host competition. The ability of larval parasitoids L. boulardi and L. heterotoma and pupal parasitoid T. cf. drosophilae to parasitize the exotic and indigenous hosts was laboratory tested. Both larval parasitoids failed to develop on D. suzukii, but high mortality was recorded in larvae exposed to L. heterotoma. On the contrary, T. cf. drosophilae developed successfully on D. suzukii, with no significant differences between the exotic and indigenous hosts. These results beg further investigations of indigenous enemies, particularly T. cf. drosophilae, for effective biological control of D. suzukii.

Biological and biochemical differences between in vitro- and in vivo-reared Exorista larvarum

Quantitative and qualitative parameters of Exorista larvarum (L.) (Diptera: Tachinidae) reared on two insect‐material‐free artificial media and in the factitious host Galleria mellonella L. (Lepidoptera: Pyralidae) were compared. Significantly higher puparial yields and weights were obtained in both a milk‐based and a veal homogenate‐based medium than in the factitious host. Longevity and parasitization rates were not different between the in vitro‐ and in vivo‐reared flies. Despite the greater puparial weight of the veal medium‐reared E. larvarum females, the number of eggs laid by these females on host larvae was not higher than that of females reared under the other two rearing conditions. Moreover, in a complementary experiment, with homogeneous puparial weights of milk medium‐ and host‐reared females, the former oviposited fewer eggs. Hence, puparial weight alone is not a reliable quality parameter for E. larvarum reared on artificial media. Lower amino acid content, with a deficiency in aromatic amino acids and an excess in proline, was found for in vitro third instar parasitoid larvae reared on both media compared to the in vivo‐reared ones. These results suggest a correlation between the amino acid deficiency and imbalance of medium‐reared larvae and the lower number of eggs laid by the females obtained.

In vitro rearing of the parasitoid Exorista larvarum (Diptera: Tachinidae) from eggs laid out of host.

We evaluated the possibility of using the eggs laid out of host to rear Exorista larvarum (L.) (Diptera: Tachinidae), a larval parasitoid of Lepidoptera, on artificial media. In a first experiment, eggs oviposited on a plastic sheet (either by inexperienced or experienced females) showed the same in vitro hatching capability as those removed from the larvae of the factitious host Galleria mellonella L. Subsequently, eggs laid on the host integument or out of host, either by inexperienced or experienced females, were removed from the oviposition substrate and placed on a skimmed milkbased artificial medium. The percentages of hatched eggs, of puparia and adults, as well as the puparial weights did not differ significantly among the three treatments. These findings suggested that E. larvarum may be successfully reared in vitro with a total exclusion of the host insect. In a further test, no difference for the in vitro hatching time was found between the eggs oviposited either on a plastic sheet or on G. mellonella larvae in the same length of time (60 min). This result suggested that at oviposition the out-of-host eggs were unincubated, similarly to those that had been laid on the host larvae.

Production of Dipteran Parasitoids

Dipteran parasitoids (especially Tachinidae, but also Sarcophagidae, Phoridae, Cryptochaetidae, and Bombyliidae) comprise a numberd of species of interest for applied biological control and, as a consequence, mass production. Although they are underestimated and often forgotten in biocontrol strategies, several studies concerning their rearing technology have been carried out. The purpose of this chapter is to review the work done on dipteran parasitoids and bring this group of insects to light. Some examples of tachinid and other dipteran parasitoids implicated in biological control are presented in the first section. Subsequently, the most important aspects of their biology relevant for rearing are described. Inxa0vivo and inxa0vitro rearing techniques are considered in another section from different points of view, such as host type (natural vs alternative) and age, infestation mode, abiotic conditions, parasitoid nutritional needs, and continuous inxa0vitro culture. Adult maintenance, quality control, and storage and shipment procedures are also discussed. Finally, some perspectives are presented with the aim of stimulating new ideas for research efforts on the mass culturing of dipteran parasitoids.

Rearing of the pupal parasitoid Brachymeria intermedia on veal homogenate-based artificial diets : evaluation of factors affecting effectiveness

The pupal parasitoid Brachymeria intermedia (Nees) was reared from egg to fecund adult on various veal homogenate‐based artificial diets. For every replicate 12.12 ml of each diet were used. Four diets were tested first. Two media, one devoid of and one supplemented with 1 ml Galleria mellonella pupal extract, contained 0.19 g wheat germ and 0.19 g yeast extract each. The other two, one added with and the other devoid of host extract, contained 0.38 g yeast extract each and no wheat germ. All diets also contained chicken egg yolk (1.1 and 0.8 ml in the diets without and with host extract, respectively). The amount of yeast extract was seen to have no significant effect on any of the developmental parameters considered. The replacement of wheat germ with yeast extract was therefore not convenient, considering that the former is far more economical than the latter. Pupal extract was instead found to have a significant effect on pupal and adult yields. The highest adult yield (= 53.2%) was obtained on the diet supplemented with 0.38 g yeast extract and containing host pupal extract. A further four media, each comprising a different kind of material derived from G. mellonella, were subsequently tested. Adult yields were such as to suggest the possibility of replacing pupal with larval extract in the diets as the latter is easier to prepare since there are no cocoons to be removed. In contrast, when the diets were supplemented with larval or pupal homogenate, adult yields dramatically dropped. When B. intermedia was reared in groups rather than individually, most larvae died before attaining maturity. Only two parasitoids, in two different replicates, emerged as adults.

Host location and suitability of the armyworm larvae of Mythimna unipuncta for the tachinid parasitoid Exorista larvarum

This work evaluates the efficiency of Exorista larvarum (L.) (Diptera: Tachinidae) in localizing and parasitizing the larvae of the noctuid moth Mythimna unipuncta (Haworth) (Lepidoptera: Noctuidae) in laboratory conditions, in a wind tunnel and in PVC cages. The most important cue in host localization by E. larvarum females was host-induced plant volatiles. Visual cues, natural maize volatiles and artificially damaged plants were less efficient for host localization. Mortality in M. unipuncta larvae on which E. larvarum females laid eggs was high. This finding indicates that E. larvarum could effectively contribute to lowering the population size of M. unipuncta, even if only few parasitoids successfully complete their development in the host. Further studies with other possible hosts must be done to apply E. larvarum successfully against M. unipuncta larvae in maize fields.

In vitro rearing of the pupal parasitoidBrachymeria intermedia (Hym.: Chalcididae) on artificial diets with and without host components

The pupal parasitoidBrachymeria intermedia (Nees) was reared from the egg to adult stage on artificial media based on commercial meat homogenates for babies (Plasmon®), either with or devoid of host components. Six media containing 80% homogenate and 20% extract ofGalleria mellonella L. pupae were tested first. Two types of homogenates, intended for babies at the beginning of weaning (a) and well on in weaning (b), were utilized. Media were based on beef, veal and chicken meat, 3 on a-homogenates and 3 on b-homogenates. A significantly higher percentage of parasitoids developed as mature larvae on the a- than on the b-homogenate based diets. This was presumably related to the higher protein, carbohydrate, lipid and calorie level of the a- than of the b- homogenates. Diet veal-a produced the best mean adult yield (27.4%). Other two diets based on the veal-a homogenate were then tested. The first, composed of 80% homogenate, 10% host pupal extract, 7% chicken egg yolk, 1.5% yeast extract and 1.5% wheat germ, produced a mean adult yield of 66.7%, similar to that obtained inG. mellonella pupae. On the second medium, devoid of host components and containing 85% veal-a homogenate, 10% chicken egg yolk, 2.5% wheat germ and 2.5% yeast extract, the mean adult yield was 22.5%. In all media, the adults obtained were viable and fecund.RésuméLe parasitoïde nymphalBrachymeria intermedia (Nees) a été élevé, de l’œuf au stade adulte, sur des milieux artificiels à base de viandes homogénéisées pour bébés, vendues dans le commerce (Plasmon®), à la fois avec ou sans matériel dérivant de l’hôte. Six milieux contenant 80% d’homogénat et 20 % d’extrait de chrysalides deGalleria mellonella L. ont d’abord été testés. Deux types d’homogénats destinés respectivement à des bébés en début de sevrage (a) et en phase de sevrage avancé (b) ont été utilisés. Pour chaque type d’homogénat, trois sortes de viande ont été testées: boeuf, veau et poulet. Les milieux à base d’homogénats de type « a » ont été plus efficaces que ceux à base d’homogénats de type « b » en ce qui concerne les pourcentages de larves en fin de développement obtenus. Cela est probablement dû au fait que les homogénats de type « a » ont une teneur plus élevée en protéines, carbohydrates, lipides et calories. Le pourcentage d’adultes le plus élevé (27,4%) a été obtenu avec le milieu à base d’homogénat de veau de type « a ». Deux milieux à base d’homogénat de veau de type « a » ont ensuite été testés. Sur le premier, contenant 80% d’homogénat, 10% d’extrait de chrysalides deG. mellonella, 75% de jaune d’oeuf, 1,5% de levure et 1,5% de germes de blé, le rendement moyen en adultes a été de 66,7%, semblable au rendement obtenu sur des chrysalides deG. mellonella. Le deuxième milieu, dépourvu de matériel dérivant de l’hôte, contenait 85% d’homogénat, 10% de jaune d’oeuf, 2,5% de germes de blé et 2,5% de levure. Le rendement obtenu sur ce milieu a été de 22,5%. Sur tous les milieux les adultes obtenus étaient viables et féconds.

Development of Aedes albopictus (Diptera: Culicidae) Larvae Under Different Laboratory Conditions

Abstract Critical to successful application of the sterile insect technique against Aedes albopictus (Skuse) is the development of an efficient and standardized rearing protocol to be employed in the mass production system. In this study, several life history traits of Ae. albopictus were analyzed to identify upper and lower thresholds of larval density and diet concentration. Survival to pupation, time to pupation, and sex ratio were evaluated under a range of larval densities (0.5–5 larvae/ml) and food levels (0.05–1.6 mg/larva/d) using two larval diets (one locally developed; one developed by the FAO/IAEA). The larvae reared at 28 °C, at a density of 2 larvae/ml and receiving a food dose equal to 0.6 mg/larva/d of a diet consisting of 50% tuna meal, 50% bovine liver powder (the FAO/ IAEA diet), and, as an additive, 0.2 g of Vitamin Mix per 100 ml of diet solution, developed in 5 d and had 90% survival to the pupal stage. With this rearing regime male pupae production 24 h after the onset of pupation was the highest; these pupae were ∼94% male.

Interactions between the Multicolored Asian Lady Beetle Harmonia axyridis and the Parasitoid Dinocampus coccinellae

Harmonia axyridis (Pallas) has been introduced either intentionally or accidentally in different areas outside its native range, where it is often regarded as invasive. Dinocampus coccinellae (Schrank) has been recorded to parasitize H. axyridis in the field, both in the native and introduced areas, Italy included. The percent of parasitism found in our field investigation was low (four percent). The effect of exposure time of H. axyridis to D. coccinellae and the impact of parasitization on host longevity, oviposition capacity and egg fertility were evaluated in the laboratory. The acceptance and suitability of H. axyridis as host for D. coccinellae were then studied, in comparison with the native coccinellid Adalia bipunctata (L.), which shares the same ecological niche. The effects of parasitization on female longevity and reproduction capacity in the exotic vs. the indigenous lady beetle were also investigated. The overall results showed that D. coccinellae negatively affected the fitness of H. axyridis, more than that of A. bipunctata. The parasitoid may thus play a marginal role in controlling the populations of the Asian lady beetle, without representing a threat to A. bipunctata.

Penetration and encapsulation of the larval endoparasitoid Exorista larvarum (Diptera: Tachinidae) in the factitious host Galleria mellonella (Lepidoptera: Pyralidae)

The tachinid fly Exorista larvarum (L.) (Diptera: Tachinidae) is a polyphagous larval endoparasitoid that deposits its eggs on the host exoskeleton of lepidopteran and tenthredinid larvae. The attachment of larval E. larvarum and the formation of the respiratory funnel were studied during infestation in the last larval instar of the wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae). The tachinid larvae burrow through the host integument after hatching, using their robust cephalopharyngeal skeleton, leaving a dark spot at the point of their penetration as a result of host cuticle melanization. Endoparasitoid penetration induces the host cellular defence, resulting in the formation of a haemocyte capsule consisting of multi-cellular sheaths. This enveloping capsule later undergoes melanization, which is mostly obvious towards the posterior part of the endoparasitoid. The endoparasitoid uses the host encapsulation response to build a respiratory funnel from the modified host integument, leading to the host surface. The encapsulated larva remains attached to the respiratory funnel via an anal hook and cuticular spines until fully developed. Additional immunohistochemical analyses were used to study host-parasitoid interactions. Indirect immunofluorescence showed no labelling of potential tachinid antigens and confirmed no effect on the surrounding host tissues. A simulated parasitization with coated polybead microspheres revealed the mortal impact of tachinid antigens to the host. Hosts injected with antigen-coated polybeads died as a consequence of an acute and extensive immunological response to the tachinid antigens and not due to the trauma caused by foreign objects inside their body.