The Susceptibility of Bemisia tabaci Mediterranean (MED) Species to Attack by a Parasitoid Wasp Changes between Two Whitefly Strains with Different Facultative Endosymbiotic Bacteria.

In this study, two strains of the mitochondrial lineage Q1 of Bemisia tabaci MED species, characterized by a different complement of facultative bacterial endosymbionts, were tested for their susceptibility to be attacked by the parasitoid wasp Eretmocerus mundus, a widespread natural enemy of B. tabaci. Notably, the BtHC strain infected with Hamiltonella and Cardinium was more resistant to parasitization than the BtHR strain infected with Hamiltonella and Rickettsia. The resistant phenotype consisted of fewer nymphs successfully parasitized (containing the parasitoid mature larva or pupa) and in a lower percentage of adult wasps emerging from parasitized nymphs. Interestingly, the resistance traits were not evident when E. mundus parasitism was compared between BtHC and BtHR using parasitoids originating from a colony maintained on BtHC. However, when we moved the parasitoid colony on BtHR and tested E. mundus after it was reared on BtHR for four and seven generations, we saw then that BtHC was less susceptible to parasitization than BtHR. On the other hand, we did not detect any difference in the parasitization of the BtHR strain between the three generations of E. mundus tested. Our findings showed that host strain is a factor affecting the ability of E. mundus to parasitize B. tabaci and lay the basis for further studies aimed at disentangling the role of the facultative endosymbiont Cardinium and of the genetic background in the resistance of B. tabaci MED to parasitoid attack. Furthermore, they highlight that counteradaptations to the variation of B. tabaci defence mechanisms may be rapidly selected in E. mundus to maximize the parasitoid fitness.

Variation among populations of Trichogramma euproctidis (Hymenoptera: Trichogrammatidae) revealed by life table parameters: perspectives for biological control.

The successful mass-rearing of potential biological control agents is a prerequisite for sustainable pest control. In this study, the performance of 3 Trichogramma euproctidis (Girault) (Hymenoptera: Trichogrammatidae) populations collected from different locations in Khuzestan (Southwest Iran) were evaluated to optimize the egg parasitoid mass-rearing for augmentative biological control of lepidopteran pests. We aimed to investigate the effects of both population origin and host quality on biological traits of ovipositing females (number of parasitized eggs) and of their progeny (development time, survival rate, sex ratio, longevity, and fecundity). The effect of host quality was assessed by allowing the parasitoid to oviposit into 1, 2, 3, or 4-day-old Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs. The 3 T. euproctidis populations developed successfully regardless the age of the host eggs. However, we found significant variation among populations and a strong influence of host quality on the traits investigated. Progeny performance in all populations decreased with increasing host age. The best-performing population (collected in Mollasani) showed the highest parasitization rate, highest survival rate, and progeny sex ratio with the greatest percentage of females. A life table corroborated these findings with superior estimates of the net reproductive rate (R0), intrinsic rate of increase (r), and reduced generation time (T) for the Mollasani population on 1-day-old host eggs. We conclude that ample variation exists among T. euproctidis populations and that rearing the Mollasani population on young rather than old eggs of E. kuehniella would be recommended to implement the biological control programs to target lepidopteran pests in Southwestern Iran.

Wolbachia infection and genetic diversity of Italian populations of Philaenus spumarius, the main vector of Xylella fastidiosa in Europe.

Philaenus spumarius is a cosmopolitan species that has become a major threat to European agriculture being recognized as the main vector of the introduced plant pathogen Xylella fastidiosa, the agent of the "olive quick decline syndrome", a disease which is devastating olive orchards in southern Italy. Wolbachia are bacterial symbionts of many insects, frequently as reproductive parasites, sometime by establishing mutualistic relationships, able to spread within host populations. Philaenus spumarius harbors Wolbachia, but the role played by this symbiont is unknown and data on the infection prevalence within host populations are limited. Here, the Wolbachia infection rate was analyzed in relation to the geographic distribution and the genetic diversity of the Italian populations of P. spumarius. Analysis of the COI gene sequences revealed a geographically structured distribution of the three main mitochondrial lineages of P. spumarius. Wolbachia was detected in half of the populations sampled in northern Italy where most individuals belonged to the western-Mediterranean lineage. All populations sampled in southern and central Italy, where the individuals of the eastern-Mediterranean lineage were largely prevalent, were uninfected. Individuals of the north-eastern lineage were found only in populations from the Alps in the northernmost part of Italy, at high altitudes. In this area, Wolbachia infection reached the highest prevalence, with no difference between north-eastern and western-Mediterranean lineage. Analysis of molecular diversity of COI sequences suggested no significant effect of Wolbachia on population genetics of P. spumarius. Using the MLST approach, six new Wolbachia sequence types were identified. Using FISH, Wolbachia were observed within the host's reproductive tissues and salivary glands. Results obtained led us to discuss the role of Wolbachia in P. spumarius, the factors influencing the geographic distribution of the infection, and the exploitation of Wolbachia for the control of the vector insect to reduce the spread of X. fastidiosa.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Biology and Prevalence in Northern Italy of Verrallia aucta (Diptera, Pipunculidae), a Parasitoid of Philaenus spumarius (Hemiptera, Aphrophoridae), the Main Vector of Xylella fastidiosa in Europe.

The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera Aphrophoridae), the main vector of Xylella fastidiosa Wells et al. in Europe, has few known natural enemies. The endoparasitoid Verrallia aucta (Fallén) (Diptera, Pipunculidae) was first noticed a long time ago but very little is known about its biology and prevalence. In this study, the presence and prevalence of V. aucta were investigated in different regions of northern Italy, both in plain-foothill and montane zones. Parasitic larvae were identified by the dissection of spittlebug adults, P. spumarius and Neophilaenus campestris (Fallén), and by a new species-specific molecular tool targeting the ITS2 and COI genomic regions, developed in this work. A small-scale rearing was set up to gain information on the life cycle of V. aucta on its main host P. spumarius. During the four-year investigation (2016-2019) the pipunculid parasitoid displayed low prevalence, reaching a maximum parasitization rate of 17.5% (calculated over the adult spittlebug season) in vineyards of the Piemonte region. Over the whole period, no significant difference in the prevalence was found between male and female spittlebugs. Collected data and rearing observations suggest that V. aucta is monovoltine and synchronous with P. spumarius, laying eggs in newly emerged adults, developing as an endoparasitoid through two larval stages during the whole summer, and overwintering as a pupa in the soil.

Electrophysiological and behavioural response of Philaenus spumarius to essential oils and aromatic plants.

The meadow spittlebug, Philaenus spumarius, is a highly polyphagous widespread species, playing a major role in the transmission of the bacterium Xylella fastidiosa subspecies pauca, the agent of the "Olive Quick Decline Syndrome". Essential oils (EOs) are an important source of bio-active volatile compounds that could interfere with basic metabolic, biochemical, physiological, and behavioural functions of insects. Here, we report the electrophysiological and behavioural responses of adult P. spumarius towards some EOs and related plants. Electroantennographic tests demonstrated that the peripheral olfactory system of P. spumarius females and males perceives volatile organic compounds present in the EOs of Pelargonium graveolens, Cymbopogon nardus and Lavandula officinalis in a dose-dependent manner. In behavioral bioassays, evaluating the adult responses towards EOs and related plants, both at close (Y-tube) and long range (wind tunnel), males and females responded differently to the same odorant. Using EOs, a clear attraction was noted only for males towards lavender EO. Conversely, plants elicited responses that varied upon the plant species, testing device and adult sex. Both lavender and geranium repelled females at any distance range. On the contrary, males were attracted by geranium and repelled by citronella. Finally, at close distance, lavender and citronella were repellent for females and males, respectively. Our results contribute to the development of innovative tools and approaches, alternative to the use of synthetic pesticides, for the sustainable control of P. spumarius aiming to contrasting the expansion of X. fastidiosa.

Zucchini Plants Alter Gene Expression and Emission of (E)-ß-Caryophyllene Following Aphis gossypii Infestation.

Zucchini (Cucurbita pepo L.) is widely cultivated in temperate regions. One of the major production challenges is the damage caused by Aphis gossypii (Homoptera: Aphididae), a polyphagous aphid, which can negatively affect its host plant, both directly by feeding and indirectly by vectoring viruses. To gain insights into the transcriptome events that occur during the zucchini-aphid interaction and to understand the early-to-late defense response through gene expression profiles, we performed RNA-sequencing (RNA-Seq) on zucchini leaves challenged by A. gossypii (24, 48, and 96 h post-infestation; hpi). Data analysis indicated a complex and dynamic pattern of gene expression and a transient transcriptional reconfiguration that involved more than 700 differentially expressed genes (DEGs), including a large number of defense-related genes. The down-regulation of key genes of plant immunity, such as leucine-rich repeat (LRR) protein kinases, transcription factors, and genes associated with direct (i.e., protease inhibitors, cysteine peptidases, etc.) and indirect (i.e., terpene synthase) defense responses, suggests the aphid ability to manipulate plant immune responses. We also investigated the emission of volatile organic compounds (VOCs) from infested plants and observed a reduced emission of (E)-ß-caryophyllene at 48 hpi, likely the result of aphid effectors, which reflects the down-regulation of two genes involved in the biosynthesis of terpenoids. We showed that (E)-ß-caryophyllene emission was modified by the duration of plant infestation and by aphid density and that this molecule highly attracts Aphidius colemani, a parasitic wasp of A. gossypii. With our results we contributed to the identification of genes involved in cucurbit plant interactions with phloem feeders. Our findings may also help pave the way toward developing tolerant zucchini varieties and to identify molecules for sustainable management of harmful insect populations.

Chlamyphilone, a Novel Pochonia chlamydosporia Metabolite with Insecticidal Activity.

Metabolites from a collection of selected fungal isolates have been screened for insecticidal activity against the aphid Acyrthosiphon pisum. Crude organic extracts of culture filtrates from six fungal isolates (Paecilomyces lilacinus, Pochonia chlamydosporia, Penicillium griseofulvum, Beauveria bassiana, Metarhizium anisopliae and Talaromyces pinophilus) caused mortality of aphids within 72 h after treatment. In this work, bioassay-guided fractionation has been used to characterize the main bioactive metabolites accumulated in fungal extracts. Leucinostatins A, B and D represent the bioactive compounds produced by P. lilacinus. From P. griseofulvum and B. bassiana extracts, griseofulvin and beauvericin have been isolated, respectively; 3-O-Methylfunicone and a mixture of destruxins have been found in the active fractions of T. pinophilum and M. anisopliae, respectively. A novel azaphilone compound, we named chlamyphilone, with significant insecticidal activity, has been isolated from the culture filtrate of P. chlamydosporia. Its structure has been determined using extensive spectroscopic methods and chemical derivatization.

Cytological analysis of cytoplasmic incompatibility induced by Cardinium suggests convergent evolution with its distant cousin Wolbachia.

Cytoplasmic incompatibility (CI) is a conditional sterility in numerous arthropods that is caused by inherited, intracellular bacteria such as Wolbachia Matings between males carrying CI-inducing Wolbachia and uninfected females, or between males and females infected with different Wolbachia strains, result in progeny that die during very early embryogenesis. Multiple studies in diploid (Drosophila) and haplodiploid (Nasonia) insects have shown that CI-Wolbachia cause a failure of the paternally derived chromatin from resolving into distinct chromosomes. This leads to the formation of chromatin bridges and other mitotic defects as early as the first mitotic division, and to early mitotic arrest. It is currently unknown if CI-inducing symbionts other than Wolbachia affect similar cellular processes. Here, we investigated CI caused by an unrelated bacterium, Cardinium, which naturally infects a parasitic wasp, Encarsia suzannae CI crosses in this host-symbiont system resulted in early mitotic defects including asynchrony of paternal and maternal chromosome sets as they enter mitosis, chromatin bridges and improper chromosome segregation that spanned across multiple mitotic divisions, triggering embryonic death through accumulated aneuploidy. We highlight small differences with CI-Wolbachia, which could be due to the underlying CI mechanism or host-specific effects. Our results suggest a convergence of CI-related cellular phenotypes between these two unrelated symbionts.

"Darwin's corollary" and cytoplasmic incompatibility induced by Cardinium may contribute to speciation in Encarsia wasps (Hymenoptera: Aphelinidae).

The potential importance of cytoplasmic incompatibility (CI)-inducing bacterial symbionts in speciation of their arthropod hosts has been debated. Theoretical advances have led to a consensus that a role is plausible when CI is combined with other isolating barriers. However, the insect model systems Nasonia and Drosophila are the only two experimental examples documented. Here, we analyzed the components of reproductive isolation between the parasitoid wasp Encarsia suzannae, which is infected by the CI-inducing symbiont Cardinium, and its uninfected sibling species Encarsia gennaroi. Laboratory crosses demonstrated that: (1) sexual isolation is incomplete; (2) hybrid offspring production is greatly reduced in the interspecific CI cross; (3) viable hybrids may be produced by curing E. suzannae males of Cardinium with antibiotics; (4) hybrid offspring production in the reciprocal cross is greatly reduced by hybrid inviability due to genetic incompatibilities; (5) hybrid sterility is nearly complete in both directions at the F1 stage. Thus, asymmetrical hybrid incompatibilities and CI act as complementary isolating mechanisms. We propose a new model for contributions of CI symbionts to speciation, with CI reducing gene flow between species in one direction, and in the other, a symbiont sweep resulting in accelerated mtDNA evolution, negative cytonuclear interactions, and hybrid incompatibilities.

Species Diversity in the Parasitoid Genus Asobara (Hymenoptera: Braconidae) from the Native Area of the Fruit Fly Pest Drosophila suzukii (Diptera: Drosophilidae).

Drosophila suzukii (Matsumura), commonly known as Spotted Wing Drosophila (SWD), is a worldwide serious economic threat to the production of berries and stone fruits. The chemical control widely used against this pest is often not able to preventing yield losses because wild flora offers an abundance of fruits to D. suzukii where the pest is able to reproduce and from where it recolonizes neighbouring cultivated fields. Alternatively, within Integrated Pest Management protocols for D. suzukii, biological control could play a key role by reducing its populations particularly in non-cultivated habitats, thus increasing the effectiveness and reducing the side negative effects of other management strategies. Because of the scarcity and of the low efficiency of autochthonous parasitoids in the new invaded territories, in the last few years, a number of surveys started in the native area of D. suzukii to find parasitoid species to be evaluated in quarantine structures and eventually released in the field, following a classical biological control approach. This paper reports the results of these surveys carried out in South Korea and for the first time in China. Among the parasitoids collected, those belonging to the genus Asobara Foerster resulted dominant both by number and species diversity. By combining morphological characters and the mitochondrial COI gene as a molecular marker, we identified seven species of Asobara, of which two associated with D. suzukii, namely A. japonica and A leveri, and five new to science, namely Asobara brevicauda, A. elongata, A mesocauda, A unicolorata, A. triangulata. Our findings offer new opportunity to find effective parasitoids to be introduced in classical biological control programmes in the territories recently invaded by D. suzukii.

Genetic Diversity of the Invasive Gall Wasp Leptocybe invasa (Hymenoptera: Eulophidae) and of its Rickettsia Endosymbiont, and Associated Sex-Ratio Differences.

The blue-gum chalcid Leptocybe invasa Fisher & LaSalle (Hymenoptera: Eulophidae) is a gall wasp pest of Eucalyptus species, likely native to Australia. Over the past 15 years it has invaded 39 countries on all continents where eucalypts are grown. The worldwide invasion of the blue gum chalcid was attributed to a single thelytokous morphospecies formally described in 2004. Subsequently, however, males have been recorded in several countries and the sex ratio of field populations has been found to be highly variable in different areas. In order to find an explanation for such sex ratio differences, populations of L. invasa from a broad geographical area were screened for the symbionts currently known as reproductive manipulators, and both wasps and symbionts were genetically characterized using multiple genes. Molecular analyses suggested that L. invasa is in fact a complex of two cryptic species involved in the rapid and efficient spread of the wasp, the first recovered from the Mediterranean region and South America, the latter from China. All screened specimens were infected by endosymbiotic bacteria belonging to the genus Rickettsia. Two closely related Rickettsia strains were found, each infecting one of the two putative cryptic species of L. invasa and associated with different average sex ratios. Rickettsia were found to be localized in the female reproductive tissues and transovarially transmitted, suggesting a possible role of Rickettsia as the causal agent of thelytokous parthenogenesis in L. invasa. Implications for the variation of sex ratio and for the management of L. invasa are discussed.

An EPG study of the probing behavior of adult Bemisia tabaci biotype Q (Hemiptera: Aleyrodidae) following exposure to cyantraniliprole.

Cyantraniliprole is a novel insecticide for control of multiple chewing and sucking insect pest species including the sweetpotato whitefly Bemisia tabaci (Gennadius), which is one of the most important polyphagous pests in tropical, subtropical, and Mediterranean regions. This study aims to evaluate the effects of cyantraniliprole on the probing behavior of B. tabaci on tomato. Electrical penetration graph data indicated that on plants treated with cyantraniliprole (foliar application), adult whiteflies of the genetic variant Q2 were not able to reach the phloem and consequently did not perform the activities represented by E1 and E2 waveforms, i.e., phloem salivation (during which inoculation of geminiviruses occurs) and phloem sap ingestion (during which geminiviruses are acquired by the whiteflies), respectively. The complete failure of B. tabaci biotype Q adults to feed from the phloem of tomato plants treated with cyantraniliprole could be explained by rapid cessation of ingestion because of the mode of action of this insecticide. Overall, these findings indicated that cyantraniliprole might represent a useful new tool for producers to protect tomato plants from damage by B. tabaci.

Invasion of the Q2 mitochondrial variant of Mediterranean Bemisia tabaci in southern Italy: possible role of bacterial endosymbionts.

BACKGROUND: The whitefly Bemisia tabaci (Gennadius) is a complex of cryptic species, some of which, namely the Mediterranean (MED) and the Middle East-Asia Minor 1 (MEAM1), are highly invasive and injurious crop pests worldwide and able to displace local genotypes. Invasiveness of B. tabaci may depend on the phenotype of inherited bacterial endosymbionts. Here, the B. tabaci genetic diversity variation that has occurred in recent years in southern Italy was examined. Whitefly was genotyped by restriction fragment length polymorphism analysis of polymerase-chain-reaction-amplified fragments (PCR-RFLP) of the COI gene and molecular identification of endosymbionts. Possible factors leading to the observed genetic diversity were examined. RESULTS: Q1 and Q2 mitochondrial types of MED, the only species found, coexisted in the field, while MEAM1 disappeared. A large spreading of Q2 (70% of individuals) was observed for the first time in Italy. Q2 showed a significant female-biased sex ratio and largely outnumbered Q1 on solanaceous hosts, in greenhouses and on insecticide-treated plants. Q1, with an even sex ratio, slightly prevailed on non-solanaceous hosts, especially on wild and untreated plants. Endosymbiont composition was associated with the mitochondrial type. Hamiltonella and Rickettsia were found at near fixation in Q1 and Q2 respectively; Arsenophonus, Cardinium and Wolbachia were found in both types, although at different frequencies. CONCLUSIONS: Q2 invasion seems to have been favoured by the agroecological conditions of southern Italy and by the female-biased sex ratio. Endosymbionts may have a role in Q2 invasiveness, acting as sex-ratio manipulators (e.g. Rickettsia) and possibly by benefiting the host fitness.