Parasitoids (Hymenoptera) of Mealybug Pests (Hemiptera: Pseudococcidae) from Southern Brazil: Molecular and Morphological Characterization.

Parasitoids of three mealybug pests (Hemiptera: Pseudococcidae), Planococcus ficus (Signoret), Pseudococcus sociabilis Hambleton, and Pseudococcus viburni (Signoret) have been identified for the first time in Brazil. Mealybugs were collected in fruit-growing areas along southern Brazil during 2013-2016. An integrative approach, combining morphological and molecular methods, was used to identify the Brazilian parasitoids to the species level. Fifteen species were recorded, including 14 primary parasitoids belonging to Encyrtidae and Platygastridae and a single secondary parasitoid species belonging to Signiphoridae. The encyrtid parasitoids Acerophagus flavidulus (Brèthes), Anagyrus calyxtoi Noyes and Zaplatycerus sp., and the signiphorid secondary parasitoid Chartocerus axillaris De Santis are reported for the first time in Brazil.

Natural enemies of armored scales (Hemiptera: Diaspididae) and soft scales (Hemiptera: Coccidae) in Chile: Molecular and morphological identification.

Scale insects (Hemiptera: Sternorrhyncha: Coccomorpha) are key pests of agricultural crops and ornamental plants worldwide. Their populations are difficult to control, even with insecticides, due to their cryptic habits. Moreover, there is growing concern over the use of synthetic pesticides for their control, due to deleterious environmental effects and the emergence of resistant populations of target pests. In this context, biological control may be an effective and sustainable approach. Hymenoptera Chalcidoidea includes natural enemies of scale insects that have been successfully used in many biological control programs. However, the correct identification of pest scale species and their natural enemies is particularly challenging because these insects are very small and highly specialized. Integrative taxonomy, coupling DNA barcoding and morphological analysis, has been successfully used to characterize pests and natural enemy species. In this study, we performed a survey of parasitoids and predators of armored and soft scales in Chile, based on 28S and COI barcodes. Fifty-three populations of Diaspididae and 79 populations of Coccidae were sampled over the entire length of the country, from Arica (18°S) to Frutillar (41°S), between January 2015 and February 2016. The phylogenetic relationships obtained by Bayesian inference from multilocus haplotypes revealed 41 putative species of Chalcidoidea, five Coccinellidae and three Neuroptera. Species delimitation was confirmed using ABGD, GMYC and PTP model. In Chalcidoidea, 23 species were identified morphologically, resulting in new COI barcodes for 12 species and new 28S barcodes for 14 species. Two predator species (Rhyzobius lophantae and Coccidophilus transandinus) were identified morphologically, and two parasitoid species, Chartocerus niger and Signiphora bifasciata, were recorded for the first time in Chile.

Investigating Biological Control Agents for Controlling Invasive Populations of the Mealybug Pseudococcus comstocki in France.

Pseudococcus comstocki (Hemiptera: Pseudococcidae) is a mealybug species native to Eastern Asia and present as an invasive pest in northern Italy and southern France since the start of the century. It infests apple and pear trees, grapevines and some ornamental trees. Biocontrol programmes against this pest proved successful in central Asia and North America in the second half of the 20th century. In this study, we investigated possible biocontrol agents against P. comstocki, with the aim of developing a biocontrol programme in France. We carried out systematic DNA-barcoding at each step in the search for a specialist parasitoid. First we characterised the French target populations of P. comstocki. We then identified the parasitoids attacking P. comstocki in France. Finally, we searched for foreign mealybug populations identified a priori as P. comstocki and surveyed their hymenopteran parasitoids. Three mealybug species (P. comstocki, P. viburni and P. cryptus) were identified during the survey, together with at least 16 different parasitoid taxa. We selected candidate biological control agent populations for use against P. comstocki in France, from the species Allotropa burrelli (Hymenoptera: Platygastridae) and Acerophagus malinus (Hymenoptera: Encyrtidae). The coupling of molecular and morphological characterisation for both pests and natural enemies facilitated the programme development and the rejection of unsuitable or generalist parasitoids.

Mealybug species from Chilean agricultural landscapes and main factors influencing the genetic structure of Pseudococcus viburni.

The present study aimed to characterize the distribution of mealybug species along Chilean agro-ecosystems and to determine the relative impact of host plant, management strategy, geography and micro-environment on shaping the distribution and genetic structure of the obscure mealybug Pseudococcus viburni. An extensive survey was completed using DNA barcoding methods to identify Chilean mealybugs to the species level. Moreover, a fine-scale study of Ps. viburni genetic diversity and population structure was carried out, genotyping 529 Ps. viburni individuals with 21 microsatellite markers. Samples from 16 localities were analyzed using Bayesian and spatially-explicit methods and the genetic dataset was confronted to host-plant, management and environmental data. Chilean crops were found to be infested by Ps. viburni, Pseudococcus meridionalis, Pseudococcus longispinus and Planococcus citri, with Ps. viburni and Ps. meridionalis showing contrasting distribution and host-plant preference patterns. Ps. viburni samples presented low genetic diversity levels but high genetic differentiation. While no significant genetic variance could be assigned to host-plant or management strategy, climate and geography were found to correlate significantly with genetic differentiation levels. The genetic characterization of Ps. viburni within Chile will contribute to future studies tracing back the origin and improving the management of this worldwide invader.

Temporal autocorrelation in host density increases establishment success of parasitoids in an experimental system.

Environmental variation is classically expected to affect negatively population growth and to increase extinction risk, and it has been identified as a major determinant of establishment failures in the field. Yet, recent theoretical investigations have shown that the structure of environmental variation and more precisely the presence of positive temporal autocorrelation might alter this prediction. This is particularly likely to affect the establishment dynamics of biological control agents in the field, as host-parasitoid interactions are expected to induce temporal autocorrelation in host abundance. In the case where parasitoid populations display overcompensatory dynamics, the presence of such positive temporal autocorrelation should increase their establishment success in a variable environment. We tested this prediction in laboratory microcosms by introducing parasitoids to hosts whose abundances were manipulated to simulate uncorrelated or positively autocorrelated variations in carrying capacity. We found that environmental variability decreased population size and increased parasitoid population variance, which is classically expected to extinction risk. However, although exposed to significant environmental variation, we found that parasitoid populations experiencing positive temporal autocorrelation in host abundance were more likely to persist than populations exposed to uncorrelated variation. These results confirm that environmental variation is a key determinant of extinction dynamics that can have counterintuitive effects depending on its autocorrelation structure.

Guiding Classical Biological Control of an Invasive Mealybug Using Integrative Taxonomy.

Delottococcus aberiae De Lotto (Hemiptera: Pseudococcidae) is a mealybug of Southern African origin that has recently been introduced into Eastern Spain. It causes severe distortions on young citrus fruits and represents a growing threat to Mediterranean citrus production. So far, biological control has proven unsatisfactory due to the absence of efficient natural enemies in Spain. Hence, the management of this pest currently relies only on chemical control. The introduction of natural enemies of D. aberiae from the native area of the pest represents a sustainable and economically viable alternative to reduce the risks linked to pesticide applications. Since biological control of mealybugs has been traditionally challenged by taxonomic misidentification, an intensive survey of Delottococcus spp. and their associated parasitoids in South Africa was required as a first step towards a classical biological control programme. Combining morphological and molecular characterization (integrative taxonomy) a total of nine mealybug species were identified in this study, including three species of Delottococcus. Different populations of D. aberiae were found on wild olive trees, in citrus orchards and on plants of Chrysanthemoides monilifera, showing intra-specific divergences according to their host plants. Interestingly, the invasive mealybug populations from Spanish orchards clustered together with the population on citrus from Limpopo Province (South Africa), sharing COI haplotypes. This result pointed to an optimum location to collect natural enemies against the invasive mealybug. A total of 14 parasitoid species were recovered from Delottococcus spp. and identified to genus and species level, by integrating morphological and molecular data. A parasitoid belonging to the genus Anagyrus, collected from D. aberiae in citrus orchards in Limpopo, is proposed here as a good biological control agent to be introduced into Spain.

Diversification of MIF immune regulators in aphids: link with agonistic and antagonistic interactions.

BACKGROUND: The widespread use of genome sequencing provided evidences for the high degree of conservation in innate immunity signalling pathways across animal phyla. However, the functioning and evolutionary history of immune-related genes remains unknown for most invertebrate species. A striking observation coming from the analysis of the pea aphid Acyrthosiphon pisum genome is the absence of important conserved genes known to be involved in the antimicrobial responses of other insects. This reduction in antibacterial immune defences is thought to be related to their long-term association with beneficial symbiotic bacteria and to facilitate symbiont maintenance. An additional possibility to avoid elimination of mutualistic symbionts is a fine-tuning of the host immune response. To explore this hypothesis we investigated the existence and potential involvement of immune regulators in aphid agonistic and antagonistic interactions. RESULTS: In contrast to the limited antibacterial arsenal, we showed that the pea aphid Acyrthosiphon pisum expresses 5 members of Macrophage Migration Inhibitory Factors (ApMIF), known to be key regulators of the innate immune response. In silico searches for MIF members in insect genomes followed by phylogenetic reconstruction suggest that evolution of MIF genes in hemipteran species has been shaped both by differential losses and serial duplications, raising the question of the functional importance of these genes in aphid immune responses. Expression analyses of ApMIFs revealed reduced expression levels in the presence, or during the establishment of secondary symbionts. By contrast, ApMIFs expression levels significantly increased upon challenge with a parasitoid or a Gram-negative bacteria. This increased expression in the presence of a pathogen/parasitoid was reduced or missing, in the presence of facultative symbiotic bacteria. CONCLUSIONS: This work provides evidence that while aphid's antibacterial arsenal is reduced, other immune genes widely absent from insect genomes are present, diversified and differentially regulated during antagonistic or agonistic interactions.

Convergent use of RhoGAP toxins by eukaryotic parasites and bacterial pathogens.

Inactivation of host Rho GTPases is a widespread strategy employed by bacterial pathogens to manipulate mammalian cellular functions and avoid immune defenses. Some bacterial toxins mimic eukaryotic Rho GTPase-activating proteins (GAPs) to inactivate mammalian GTPases, probably as a result of evolutionary convergence. An intriguing question remains whether eukaryotic pathogens or parasites may use endogenous GAPs as immune-suppressive toxins to target the same key genes as bacterial pathogens. Interestingly, a RhoGAP domain-containing protein, LbGAP, was recently characterized from the parasitoid wasp Leptopilina boulardi, and shown to protect parasitoid eggs from the immune response of Drosophila host larvae. We demonstrate here that LbGAP has structural characteristics of eukaryotic RhoGAPs but that it acts similarly to bacterial RhoGAP toxins in mammals. First, we show by immunocytochemistry that LbGAP enters Drosophila immune cells, plasmatocytes and lamellocytes, and that morphological changes in lamellocytes are correlated with the quantity of LbGAP they contain. Demonstration that LbGAP displays a GAP activity and specifically interacts with the active, GTP-bound form of the two Drosophila Rho GTPases Rac1 and Rac2, both required for successful encapsulation of Leptopilina eggs, was then achieved using biochemical tests, yeast two-hybrid analysis, and GST pull-down assays. In addition, we show that the overall structure of LbGAP is similar to that of eukaryotic RhoGAP domains, and we identify distinct residues involved in its interaction with Rac GTPases. Altogether, these results show that eukaryotic parasites can use endogenous RhoGAPs as virulence factors and that despite their differences in sequence and structure, eukaryotic and bacterial RhoGAP toxins are similarly used to target the same immune pathways in insects and mammals.