Parasitic dodder expresses an arsenal of secreted cellulases with multi-substrate specificity during host invasion.

Cuscuta campestris is a common and problematic parasitic plant which relies on haustoria to connect to and siphon nutrients from host plants. Glycoside hydrolase family 9 (GH9) cellulases (EC 3.2.1.4) play critical roles in plant cell wall biosynthesis and disassembly, but their roles during Cuscuta host invasion remains underexplored. In this study, we identified 22 full-length GH9 cellulase genes in C. campestris genome, which encoded fifteen secreted and seven membrane-anchored cellulases that showed distinct phylogenetic relationships. Expression profiles suggested that some of the genes are involved in biosynthesis and remodeling of the parasite's cell wall during haustoriogenesis, while other genes encoding secreted B- and C-type cellulases are tentatively associated with degrading host cell walls during invasion. Transcriptomic data in a host-free system and in the presence of susceptible or partially resistant tomato hosts, showed for especially GH9B7, GH9B11 and GH9B12 a shift in expression profiles in the presence of hosts, being more highly expressed during host attachment, indicating that Cuscuta can tune cellulase expression in response to a host. Functional analyses of recombinant B- and C-type cellulases showed endoglucanase activities over wide pH and temperature conditions, and activities towards multiple cellulose and hemicellulose substrates. These findings improve our understanding of host cell wall disassembly by Cuscuta, and cellulase activity towards broad substrate range potentially explain its wide host range. This is the first study to provide a broad biochemical insight into Cuscuta GH9 cellulases, which based on our study may have potential applications in industrial bioprocessing.

Rising from the shadows: Selective foraging in model shoot parasitic plants.

Despite being sessile, plants nonetheless forage for resources by modulating their growth. Adaptative foraging in response to changes in resource availability and presence of neighbours has strong implications for performance and fitness. It is an even more pressing issue for parasitic plants, which draw resources directly from other plants. Indeed, parasitic plants were demonstrated over the years to direct their growth towards preferred hosts and invest resources in parasitism relative to host quality. In contrast to root parasites that rely mostly on chemical cues, some shoot parasites seem to profit from the ability to integrate different types of abiotic and biotic cues. While significant progress in this field has been made recently, there are still many open questions regarding the molecular perception and the integration of diverse signalling pathways under different ecological contexts. Addressing how different cues are integrated in parasitic plants will be important when unravelling variations in plant interaction pathways, and essential to predict the spread of parasites in natural and agricultural environments. In this review, we discuss this with a focus on Cuscuta species as an emerging parasitic model, and provide research perspectives based on the recent advances in the topic and plant-plant interactions in general.

Cuscuta campestris fine-tunes gene expression during haustoriogenesis as an adaptation to different hosts.

The Cuscuta genus comprises obligate parasitic plants that have an unusually wide host range. Whether Cuscuta uses different infection strategies for different hosts or whether the infection strategy is mechanistically and enzymatically conserved remains unknown. To address this, we investigated molecular events during the interaction between field dodder (Cuscuta campestris) and two host species of the Solanum genus that are known to react differently to parasitic infection. We found that host gene induction, particularly of cell wall fortifying genes, coincided with a differential induction of genes for cell wall degradation in the parasite in the cultivated tomato (Solanum lycopersicum) but not in a wild relative (Solanum pennellii). This indicates that the parasite can adjust its gene expression in response to its host. This idea was supported by the increased expression of C. campestris genes encoding an endo-ß-1,4-mannanase in response to exposure of the parasite to purified mono- and polysaccharides in a host-independent infection system. Our results suggest multiple key roles of the host cell wall in determining the outcome of an infection attempt.

A host-free transcriptome for haustoriogenesis in Cuscuta campestris: Signature gene expression identifies markers of successive development stages.

The development of the infection organ of the parasitic angiosperm genus Cuscuta is a dynamic process that is normally obscured from view as it happens endophytically in its host. We artificially induced haustoriogenesis in Cuscuta campestris by far-red light to define specific morphologically different stages and analyze their transcriptional patterns. This information enabled us to extract sets of high-confidence housekeeping and marker genes for the different stages, validated in a natural infection setting on a compatible host. This study provides a framework for more reproducible investigations of haustoriogenesis and the processes governing host-parasite interactions in shoot parasites, with C. campestris as a model species.

Direct and Indirect Effect via Endophytism of Entomopathogenic Fungi on the Fitness of Myzus persicae and Its Ability to Spread PLRV on Tobacco.

Aphids are major crop pests that transmit more than half of all insect-vectored plant viruses responsible for high yield losses worldwide. Entomopathogenic fungi (EPF) are biological control agents mainly used by foliar application to control herbivores, including sap-sucking pests such as aphids. Their ability to colonize plant tissues and to interact with diverse plant pathogenic microorganisms have been reported. In our study, we evaluated the effectiveness of Beauveria bassiana ((Balsamo-Crivelli) Vuillemin) directly applied by contact or/and indirectly via endophytism in tobacco plants (Nicotiana tabacum L.) against the virus vector Myzus persicae (Sulzer) carrying the Potato leafroll virus (PLRV) or not. We found that both contact treatment and endophytic colonization of leaves significantly increased aphid mortality and decreased the fecundity rate when compared to control plants. In addition, on fungal-colonized leaves, viruliferous aphids were more negatively impacted than virus-free ones and nymph mortality was significantly higher than on fungal-free plants. Furthermore, we assessed PLRV transmission by M. persicae on tobacco plants inoculated with either B. bassiana or Metarhizium acridum ((Driver and Milner) JF Bischoff, Rehner, and Humber) as source or/and recipient plants. Myzus persicae was found to acquire and transmit PLRV regardless of the treatment. Nevertheless, the infection rate of endophytically colonized plants was lower at a seven-day incubation period and had increased to almost 100% after fifteen days. These results suggest that B. bassiana is effective against aphids, both by contact and via endophytism, and both B. bassiana and M. acridum delayed PLRV infection in tobacco.

Does the Infectious Status of Aphids Influence Their Preference Towards Healthy, Virus-Infected and Endophytically Colonized Plants?

Aphids (Hemiptera: Aphididae) cause significant damage and transmit viruses to various crop plants. We aimed to evaluate how the infectious status of aphids influences their interaction with potential hosts. Two aphid (Myzus persicae and Rhopalosiphum padi) and plant (Nicotiana tabacum and Triticum aestivum) species were used. The preferences of aphids towards healthy, virus-infected (Potato Leafroll Virus (PLRV) and Barley Yellow Dwarf virus (BYDV)), and endophytic entomopathogenic fungi (EEPF)-inoculated (Beauveria bassiana and Metarhizium acridum) plants were investigated in dual-choice tests. The headspace volatiles of the different plant modalities were also sampled and analyzed. Viruliferous and non-viruliferous aphids were more attracted to EEPF-inoculated plants compared to uninoculated plants. However, viruliferous aphids were more attracted to EEPF-inoculated plants compared to virus-infected plants, while non-viruliferous insects exhibited no preference. Fungal-inoculated plants released higher amounts of aldehydes (i.e., heptanal, octanal, nonanal and decanal) compared to other plants, which might explain why viruliferous and non-viruliferous aphids were more abundant in EEPF-inoculated plants. Our study provides an interesting research perspective on how EEPF are involved in behavior of virus vector, depending on the infectious status of the latter.

Oviposition deterrent activity of basil plants and their essentials oils against Tuta absoluta (Lepidoptera: Gelechiidae).

The leafminer Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) is one of the most important pests of tomato, reducing crop yields by up to 100% in greenhouses and fields, in several countries globally. Because synthetic insecticides lead to resistance and have adverse effects on natural enemies and the health of producers, alternative control methods are needed. In this study, we assessed the oviposition-deterring effect of basil plants, Ocimum gratissimum L. and O. basilicum L. (Lamiaceae), using dual-choice behavioural assays performed in flight tunnels. We found that both plants significantly reduced T. absoluta oviposition behaviour on a tomato plant located nearby. To evaluate the potential effect of basil volatile organic compounds, we formulated essential oils of both plant species in paraffin oil, and observed a similar oviposition-deterring effect. Gas chromatography analyses detected 18 constituents in these essential oils which the major constituents included thymol (33.3%), p-cymene (20.4%), γ-terpinene (16.9%), myrcene (3.9%) in O. gratissimum and estragol (73.8%), linalool (8.6%), ß-elemene (2.9%) and E-ß-ocimene (2.6%) in O. basilicum. Twenty and 33 compounds were identified of the volatiles collected on O. gratissimum and O. basilicum plants, respectively. The main components include the following: p-cymene (33.5%), γ-terpinene (23.6%), α-terpinene (7.2%), α-thujene (6.7%) and E-α-bergamotene (38.9%) in O. gratissimum, and methyl eugenol (26.1%), E-ß-ocimene (17.7%), and linalool (9.4%) in O. basilicum. Four compounds (α-pinene, ß-pinene, Myrcene, Limonene) were common in essential oils and plants. Our results suggest the valuable potential of basil and associated essential oils as a component of integrated management strategies against the tomato leafminer.

Structure and distribution of the sensilla on the antennae of Tuta absoluta (Lepidoptera: Gelechiidae).

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread devastating pest that develops on tomato and other economically important solanaceous crops. Current semiochemically-based management strategies still fail to significantly reduce damages and need to be improved. Here we describe under scanning and transmission electron microscopy the structure and distribution of the sensilla that are displayed on adult antennae. These were similar in size between males (3424.4±135.3µm) and females (3292.1±111.5µm), being segmented into a scape, a pedicel, and a distal filiform flagellum. Eight morphological sensilla types were observed on both sexes: Böhm's bristles, sensilla squamiformia, sensilla trichodea, sensilla basiconica (two subtypes), sensilla chaetica, sensilla coeloconica, sensilla auricillica, and sensilla styloconica. The main sexual dimorphism was related to the higher abundance of sensilla trichodea in males, twice as abundant as in females. The putative functional significance of the different sensilla types regarding the insect ecology is discussed based on the available literature. This work provides descriptions of the antennae and related sensory structures. We expect these results to help develop further electrophysiological investigations aiming to a better understanding of T. absoluta olfaction.

Histopathological effects of Aspergillus clavatus (Ascomycota: Trichocomaceae) on larvae of the southern house mosquito, Culex quinquefasciatus (Diptera: Culicidae).

Aspergillus clavatus (Ascomycota: Trichocomaceae) was previously found to be an opportunistic pathogen of mosquitoes (Diptera: Culicidae). In the present study, the mechanism leading to its insecticidal activity was investigated regarding histological damages on Culex quinquefasciatus larvae exposed to A. clavatus spores. Multiple concentration assays using spore suspensions (0.5-2.5 × 10(8) spores ml(-1)) revealed 17.0-74.3 % corrected mortalities after 48 h exposure. Heat-deactivated spores induced a lower mortality compared to nonheated spores suggesting that insecticidal effects are actively exerted. Spore-treated and untreated larvae were prepared for light microscopy as well as for scanning and transmission electron microscopy. Spores failed to adhere to the external body surface (except the mouth parts) of these aquatic immature stages but progressively filled the digestive tract where their metabolism seemed to activate. In parallel, the internal tissues of the larvae, i.e. the midgut wall, the skeletal muscles, and the cuticle-secreting epidermis, were progressively destroyed between 8 and 24 h of exposure. These observations suggest that toxins secreted by active germinating spores of A. clavatus in the digestive tract altered the larval tissues, leading to their necrosis and causing larval death. Fungal proliferation and sporulation then occurred during a saprophytic phase. A. clavatus enzymes or toxins responsible for these pathogenic effects need to be identified in further studies before any use of this fungus in mosquito control.

Diversity and ecology survey of mosquitoes potential vectors in Belgian equestrian farms: A threat prevention of mosquito-borne equine arboviruses.

Emergence of West Nile Virus was recently recorded in several European countries, which can lead to severe health problems in horse populations. Europe is also at risk of introduction of mosquito-borne equine alphavirus from Americas. Prevention of these arboviruses requires a clear understanding of transmission cycles, especially their vectors. To characterize mosquito fauna, their ecology and identify potential vectors of equine arboviruses in Belgium, entomological surveys of six equestrian farms located in the Wolloon Region were conducted during 2011-2012. The harvest of mosquitoes was based on larval sampling (272 samples from 111 breeding sites) and monthly adults trapping (CO2-baited traps, Mosquito Magnet Liberty Plus). Among 51,493 larvae and 319 adult mosquitoes collected, morphological identification showed the presence of 11 species: Anopheles claviger (Meigen), An. maculipennis s.l. (Meigen), An. plumbeus (Stephens), Culex hortensis (Ficalbi), Cx. territans (Walker), Cx. pipiens s.l. L., Cx. torrentium (Martini), Coquillettidia richiardii (Ficalbi), Culiseta annulata (Schrank), Aedes cantans (Meigen), Ae. geniculatus (Olivier). Molecular identification of Cx. pipiens species complex allowed the detection of three molecular forms, Pipiens (92.3%), Molestus (4.6%) and Hybrid (3.1%). Larvae of Cx. pipiens sl and Cx. torrentium were omnipresent and the most abundant species. Water troughs, ponds and slurry (liquid manure) were the most favorable breeding sites of mosquito larvae. Based upon behavior and ecology of the identified mosquito species, Studied Belgian equestrian farms seem to provide a suitable environment and breeding sites for the proliferation of potential vectors of arboviruses and those being a real nuisance problem for horses and neighboring inhabitants.

Infestation Level Influences Oviposition Site Selection in the Tomato Leafminer Tuta absoluta (Lepidoptera: Gelechiidae).

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a devastating pest that develops principally on solanaceous plants throughout South and Central America and Europe. In this study, we tested the influence of three levels of T. absoluta infestations on the attraction and oviposition preference of adult T. absoluta. Three infestation levels (i.e., non-infested plants, plants infested with 10 T. absoluta larvae, and plants infested with 20 T. absoluta larvae) were presented by pairs in a flying tunnel to groups of T. absoluta adults. We found no differences in terms of adult attraction for either level of infestations. However, female oviposition choice is influenced by larvae density on tomato plants. We discuss the underlying mechanisms and propose recommendations for further research.

Reintroduction of the invasive mosquito species Aedes albopictus in Belgium in July 2013.

Since its first report in 2000, the invasive mosquito Aedes albopictus was not found any more during the different entomological inspections performed at its place of introduction in Belgium between 2001 and 2012. In July 2013, one adult male was captured at the same site (a platform of imported used tires located in Vrasene, Oost-Vlaanderen Province), during a monitoring using CO2-baited trap. This finding suggests the reintroduction of the species in Belgium via the used tire trade.