Beneficial effect of supplementing an artificial diet for Amblyseius swirskii with Hermetia illucens haemolymph

Artificial diets have been developed to sustain the mass rearing of a wide range of arthropod natural enemies, with varying success. In some cases, such diets can be optimized using insect‐derived materials, such as haemolymph. In this study, we examined the effect of supplementing haemolymph of the black soldier fly, Hermetia illucens, to a basic artificial diet for the phytoseiid mite Amblyseius swirskii. The survival, development and reproduction of the predatory mite were assessed when fed on artificial diets composed of honey, sucrose, tryptone, yeast extract and egg yolk, supplemented with 5%, 10%, or 20% of H. illucens pre‐pupal haemolymph. Developmental time from larva to adult was shorter for males and females offered artificial diets supplemented with 20% haemolymph vs. the basic diet. The oviposition rate and total fecundity of females reared on the basic diet were substantially lower than those of females supplied with the enriched diets. The intrinsic rate of increase was highest on the diet containing 20% haemolymph, followed by those containing 10% and 5% haemolymph. In a subsequent diet‐switching experiment, mites fed on the basic diet in their juvenile stages were switched upon adulthood to diet enriched with different concentrations of H. illucens haemolymph. The females that were fed with the enriched diets from the adult stage on had higher oviposition rates and fecundities than those maintained on the basic diet, but their reproductive parameters were not significantly affected by the concentration of the haemolymph in the artificial diet. In conclusion, supplementing artificial diets with black soldier fly haemolymph significantly improved their nutritional value for A. swirskii. Our findings indicate the potential of using H. illucens as a cheap source for haemolymph in artificial diets, as the fly can be cost‐effectively produced at a large scale on organic waste materials.     

Acaricides impair prey location in a predatory phytoseiid mite

The use of predatory mites as the sole management tactic in biological control programmes frequently does not fully and reliably prevents damage of phytophagous mites on plants. Therefore, as an alternative, the integration of predatory mites with acaricides can provide more effective control of phytophagous mites than that of the predators only. However, for such integration, acaricides minimal negative impacts on predatory mites are required. In this study, we evaluated the sublethal effects of three acaricides on the foraging behaviour of Neoseiulus baraki (Athias‐Henriot) (Acari: Phytoseiidae) in a coconut production system. The acaricides were assessed for interference with the location of prey habitat using a Y‐tube olfactometer and for interference with the location of the prey colony within the habitat using a video‐tracking system. In addition to the choice of odour source, the time required and the distance walked to make the choice were assessed. The acaricides tested were abamectin, azadirachtin and fenpyroximate. The predatory mite preferred coconuts infested with the coconut mite Aceria guerreronis Keifer (Acari: Eriophyidae) over uninfested coconuts when not exposed to acaricides. However, when exposed to acaricides, the predator did not distinguish between infested and uninfested fruits. When exposed to abamectin, N. baraki spent more time resting and walked greater distances before making the choice of an odour source. Thus, the acaricides impair the ability of the predatory mite N. baraki to locate a prey habitat and to locate a prey within that habitat. The acaricides differentially affected prey foraging by interfering with odour perception.     

Honey bee colonies from different races show variation in defenses against the varroa mite in a ‘common garden’

Honey bee [Apis mellifera L. (Hymenoptera: Apidae)] genetic diversity may be the key to responding to novel health challenges faced by this important pollinator. In this study, we first compared colonies of four honey bee races, A. m. anatoliaca, A. m. carnica, A. m. caucasica, and A. m. syriaca from Turkey, with respect to honey storage, bee population size, and defenses against varroa. The mite Varroa destructor Anderson & Trueman (Acari: Varroidae) is an important pest of honey bee colonies. There are genetic correlates with two main defenses of bees against this parasite: hygienic behavior, or removing infested brood, and grooming, which involves shaking and swiping off mites and biting them. In the second part of this study, we examined the relationship of these two types of defenses, hygiene and grooming, and their correlation with infestation rates in 32 genetically diverse colonies in a ‘common garden’ apiary. Mite biting was found to be negatively correlated with mite infestation levels.     

Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato

Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro‐based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone‐ related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis‐related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya.     

Disentangling mite predator‐prey relationships by multiplex PCR

Gut content analysis using molecular techniques can help elucidate predator‐prey relationships in situations in which other methodologies are not feasible, such as in the case of trophic interactions between minute species such as mites. We designed species‐specific primers for a mite community occurring in Spanish citrus orchards comprising two herbivores, the Tetranychidae Tetranychus urticae and Panonychus citri, and six predatory mites belonging to the Phytoseiidae family; these predatory mites are considered to be these herbivores’ main biological control agents. These primers were successfully multiplexed in a single PCR to test the range of predators feeding on each of the two prey species. We estimated prey DNA detectability success over time (DS₅₀), which depended on the predator‐prey combination and ranged from 0.2 to 18 h. These values were further used to weight prey detection in field samples to disentangle the predatory role played by the most abundant predators (i.e. Euseius stipulatus and Phytoseiulus persimilis). The corrected predation value for E. stipulatus was significantly higher than for P. persimilis. However, because this 1.5‐fold difference was less than that observed regarding their sevenfold difference in abundance, we conclude that P. persimilis is the most effective predator in the system; it preyed on tetranychids almost five times more frequently than E. stipulatus did. The present results demonstrate that molecular tools are appropriate to unravel predator‐prey interactions in tiny species such as mites, which include important agricultural pests and their predators.     

Role of Arginine decarboxylase (ADC) in Arabidopsis thaliana defence against the pathogenic bacterium Pseudomonas viridiflava

Polyamine biosynthesis starts with putrescine production through the decarboxylation of arginine or ornithine. In Arabidopsis thaliana, putrescine is synthesised exclusively by arginine decarboxylase (ADC), which exists as two isoforms (ADC1 and 2) that are differentially regulated by abiotic stimuli, but their role in defence against pathogens has not been studied in depth. This work analysed the participation of ADC in Arabidopsis defence against Pseudomonas viridiflava. ADC activity and expression, polyamine levels and bacterial resistance were analysed in null mutants of each ADC isoform. In non‐infected wild‐type (WT) plants, ADC2 expression was much higher than ADC1. Analysis of adc mutants demonstrated that ADC2 contributes to a much higher extent than ADC1 to basal ADC activity and putrescine biosynthesis. In addition, adc2 mutants showed increased basal expression of salicylic acid‐ and jasmonic acid‐dependent PR genes. Bacterial infection induced putrescine accumulation and ADC1 expression in WT plants, but pathogen‐induced putrescine accumulation was blocked in adc1 mutants. Results suggest a specific participation of ADC1 in defence, although basal resistance was not decreased by dysfunction of either of the two ADC genes. In addition, and as opposed to WT plants, bacterial infection increased ADC2 expression and ADC activity in adc1 mutants, which could counterbalance the lack of ADC1. Results demonstrate a major contribution of ADC2 to total ADC activity and the specific induction of ADC1 in response to infection. A certain degree of functional redundancy between the two isoforms in relation to their contribution to basal resistance is also evident.     

Mix‐planting pubescent and glabrous cassava affects abundance of Typhlodromalus aripo and its prey mite Mononychellus tanajoa

There is an increasing awareness that vegetation diversity can affect herbivore and natural enemy abundance and that plants can play a major role in directly manipulating natural enemy abundance for protection against herbivore attacks. Using data from cassava fields, we aimed at (i) testing the capacity of the predatory mite Typhlodromalus aripo to control the herbivorous mite Mononychellus tanajoa in a chemical exclusion trial; and (ii) testing, based on the differential preference by T. aripo for cassava cultivars, how combinations of two morphologically different cassava cultivars with differential suitability to the predator can improve its population densities on the non‐favourable cultivar, thereby reducing M. tanajoa densities with subsequent increases in cassava yield. The study was conducted in a cassava field in Benin, West Africa. The experiments confirmed that T. aripo effectively suppresses M. tanajoa populations on both cultivars and showed, in the no‐predator‐exclusion experiments, that cultivar combinations have significant effects on M. tanajoa and T. aripo densities. Indeed, T. aripo load on the non‐preferred cultivar was lowest in subplots where the proportion of T. aripo‐preferred cultivar was also low, while, and as expected, M. tanajoa load on the non‐preferred cultivar showed decreasing trends with increasing T. aripo densities. The possible mechanisms by which cultivar mixing could increase predator load on the non‐favourable cultivar were discussed. Our data showed that appropriate cultivar combinations effectively compensate for morphologically related differences in natural enemy abundance on a normally predator‐deficient cultivar, resulting in lower pest densities on the non‐favourable cultivar. In practical terms, this strategy could, in part, enhance adoption of cultivars that do not support sufficient levels of natural enemies for pest control.     

A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

Grapevine VpPR10.1 functions in resistance to Plasmopara viticola through triggering a cell death‐like defence response by interacting with VpVDAC3

As one of the most serious diseases in grape, downy mildew caused by Plasmopara viticola is a worldwide grape disease. Much effort has been focused on improving susceptible grapevine resistance, and wild resistant grapevine species are important for germplasm improvement of commercial cultivars. Using yeast two‐hybrid screen followed by a series of immunoprecipitation experiments, we identified voltage‐dependent anion channel 3 (VDAC3) protein from Vitis piasezkii ‘Liuba‐8’ as an interacting partner of VpPR10.1 cloned from Vitis pseudoreticulata ‘Baihe‐35‐1’, which is an important germplasm for its resistance to a range of pathogens. Co‐expression of VpPR10.1/VpVDAC3 induced cell death in Nicotiana benthamiana, which accompanied by ROS accumulation. VpPR10.1 transgenic grapevine line showed resistance to P. viticola. We conclude that the VpPR10.1/VpVDAC3 complex is responsible for cell death‐mediated defence response to P. viticola in grapevine.     

Unrelated facultative endosymbionts protect aphids against a fungal pathogen

The importance of microbial facultative endosymbionts to insects is increasingly being recognized, but our understanding of how the fitness effects of infection are distributed across symbiont taxa is limited. In the pea aphid, some of the seven known species of facultative symbionts influence their host's resistance to natural enemies, including parasitoid wasps and a pathogenic fungus. Here we show that protection against this entomopathogen, Pandora neoaphidis, can be conferred by strains of four distantly related symbionts (in the genera Regiella, Rickettsia, Rickettsiella and Spiroplasma). They reduce mortality and also decrease fungal sporulation on dead aphids which may help protect nearby genetically identical insects. Pea aphids thus obtain protection from natural enemies through association with a wider range of microbial associates than has previously been thought. Providing resistance against natural enemies appears to be a particularly common way for facultative endosymbionts to increase in frequency within host populations.     

Varroa treatment with bromfenvinphos markedly suppresses honeybee biochemical defence levels

We examined the influence of bromfenvinphos, a commonly used acaricide, on activities of many metabolic enzymes affecting the biochemical defences/physiology of the western honeybee, Apis mellifera L. (Hymenoptera: Apidae), as well as on some metabolic compound concentrations, percentage of global DNA methylation, and Nosema spp. infection levels. Bromfenvinphos‐treated workers had decreased haemolymph volumes and higher protein concentrations on their cuticle but lower protein concentrations in the haemolymph. They had higher global DNA methylation levels independent of the age‐related variants. Bromfenvinphos decreased the activities of antioxidant enzymes (SOD, GPx, CAT, GST), acidic, neutral, and alkaline protease inhibitors and enzymatic physiological markers (AST, ALT, ALP), and concentrations of urea, uric acid, creatinine, cholesterol, glucose, Mg²⁺, and Ca²⁺ in worker haemolymph, depending on the age of the bees. Protease activities were higher only in the haemolymph of young bromfenvinphos‐treated bees in comparison with untreated bees. This compound decreased the activities of alkaline proteases and neutral protease inhibitors on the cuticle. Unexpectedly, in the treated bees, the activities of acidic and neutral proteases, and acidic and alkaline protease inhibitors, were higher in the young bees and lower in the older workers in comparison to the untreated group. The bromfenvinphos‐treated workers were more heavily infested with Nosema spp. Thus, bromfenvinphos not only supressed many levels of biochemical defences, and therefore stress‐resistance‐related biochemical pathways but also visibly increased the Nosema spp. infection levels.     

The reproductive capability of Ooencyrtus kuvanae reared on eggs of the factitious host Antheraea pernyi

Ooencyrtus kuvanae is a key egg parasitoid of Lymantria dispar (L.), an important defoliating pest in China and North America. We have developed mass rearing techniques for O. kuvanae and in this study compared the reproductive capacity of O. kuvanae when it was previously reared on the natural host, L. dispar or the factitious host, Antheraea pernyi Guerin‐Meneville. There was no significant difference in the oviposition period or total number of eggs laid between L. dispar‐reared and A. pernyi‐reared females. However, the mean number of offspring successfully emerging from those eggs was significantly larger from the A. pernyi‐reared females compared with L. dispar‐reared females. From this, we can conclude that, with respect to reproductive capability, eggs of A. pernyi are suitable factitious hosts for mass rearing of O. kuvanae.     

Limited dispersal in an ectoparasitic mite,Laelaps giganteus,contributes to significant phylogeographic congruence with the rodent host,Rhabdomys

To explore how biogeography, parasite life history and host vagility influences evolutionary codivergences, we followed a comparative phylogeography approach using a host‐specific nonpermanent mite, Laelaps giganteus, that occurs on four rodent species within the genus Rhabdomys. A mtDNA COI haplotype network derived for 278 parasite specimens showed marked phylogeographic congruence with host distributions. Analysis of the less variable nuclear intron Tropomyosin was in part consistent with these results. Although distance‐based cophylogenetic analyses in axparafit failed to support significant mtDNA codivergences (P ≥ 0.02), event‐based analyses revealed significant cophylogeny between sampling localities of Rhabdomys and Laelaps using core‐pa (P = 0.046) and jane (P = 0.026; P = 0.00). These findings, in conjunction with the weak congruence previously reported among the permanent ectoparasitic lice Polyplax and Rhabdomys, suggest that host–parasite intimacy is not the most important driver of significant codivergence in our study system. Instead, the more restricted dispersal ability of L. giganteus, when compared to Polyplax, resulted in stronger spatial structuring and this could have resulted in significant codivergence. Host switching occurred predominantly on the edges of host distributions and was probably facilitated by climate‐induced range shifts. When host ranges shift, the phylogeographic structure of L. giganteus is not reflecting the host movements as most of the nest bound parasites do not disperse with the host (they miss the boat) and the genetic contribution of the few dispersing mite individuals is often overwhelmed by the large number of individuals already present in nests within the new environment (causing them to drown on arrival).     

Accumulation patterns of endogenous β‐aminobutyric acid during plant development and defence in Arabidopsis thaliana

• We recently discovered that β‐aminobutyric acid (BABA), a molecule known for its ability to prime defences in plants, is a natural plant metabolite. However, the role played by endogenous BABA in plants is currently unknown. In this study we investigated the systemic accumulation of BABA during pathogen infection, levels of BABA during plant growth and development and analysed mutants possibly involved in BABA transport or regulation.
• BABA was quantified by LC‐MS using an improved method adapted from a previously published protocol. Systemic accumulation of BABA was determined by analysing non‐infected leaves and roots after localised infections with Plectosphaerella cucumerina or Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpt2. The levels of BABA were also quantified in different plant tissues and organs during normal plant growth, and in leaves during senescence. Mutants affecting amino acid transport (aap6, aap3, prot1 and gat1), γ‐aminobutyric acid levels (pop2) and senescence/defence (cpr5‐2) were analysed.
• BABA was found to accumulate only locally after bacterial or fungal infection, with no detectable increase in non‐infected systemic plant parts. In leaves, BABA content increased during natural and induced senescence. Reproductive organs had the highest levels of BABA, and the mutant cpr5‐2 produced constitutively high levels of BABA.
• Synthetic BABA is highly mobile in the receiving plant, whereas endogenous BABA appears to be produced and accumulated locally in a tissue‐specific way. We discuss a possible role for BABA in age‐related resistance and propose a comprehensive model for endogenous and synthetic BABA.

     

Bacterial iturins mediate biocontrol activity of Bacillus sp. against postharvest pear fruit‐rotting fungi

A potential antagonist, Bacillus sp. LYLB4 isolated from pear fruits, was tested for its antifungal activity against postharvest pear pathogens. LYLB4 had a remarkable antifungal effect on Botryosphaeria dothidea. Although it showed a weak inhibition effect on the growth of Rhizopus stolonifer on potato dextrose agar (PDA) plates, LYLB4 almost completely destroyed R. stolonifer during direct contact in potato dextrose broth (PDB). LYLB4 treatment was able to significantly reduce disease incidence (by 68.9% and 100%, respectively) and lesion diameter (by 68.7% and 100%, respectively) of ring rot caused by B. dothidea and soft rot caused by R. stolonifer in pears. LYLB4 also suppressed several other phytopathogens in vitro, suggesting a broad‐spectrum antagonistic activity against fungal pathogens. 16S rRNA and gyrA sequence analysis indicated that LYLB4 is closely related to B. velezensis. Genome mining indicated that LYLB4 had 11 secondary metabolites encoding clusters, but that the surfactin and fengycin gene clusters may not be functional because of a large deletion. Matrix‐assisted laser desorption ionization‐time of flight mass spectra (MALDI‐TOF‐MS) demonstrated that iturins were the major lipopeptides, and that C₁₆/C₁₇ Bacillomycin D synthesis was stimulated when LYLB4 was co‐cultured with B. dothidea compared to the control. Overall, these results demonstrate that the main biocontrol mechanism adopted by LYLB4 could be through the production of toxic metabolites and direct contact with pathogens.     

Heat curing Metaseiulus occidentalis (Nesbitt) (Acari,Phytoseiidae) of a fitness-reducing microsporidium

Laboratory colonies of the predatory mite Metaseiulus occidentalis in Gainesville, FL were found to be infected with an undescribed microsporidium. Experiments were performed to quantify the effect of infection on the fitness of M. occidentalis and to determine if heat treatment can cure mites of the microsporidium. The colonies tested were derived from an isofemale line so that differences in performance could be attributed to the presence of microsporidia. A subcolony of an uninfected isofemale line was infected with the microsporidium by feeding females infected eggs from another colony of M. occidentalis. Infected mites had a shorter mean (+/-SD) female life span (7.4 +/- 2.9 vs. 10.0 +/- 2.8 days), lower mean oviposition (1.6 +/- 0.7 vs. 2.2 +/- 0.4 eggs/day), and a male-biased sex ratio (43 +/- 16% vs. 57 +/- 15% female progeny). The infection was reduced temporarily in colonies initiated from mites that were reared in a growth chamber at 33 degrees C from egg to adult, but healthy colonies only were established from the progeny of the heat-treated adults. These colonies remained free of infection for 10 weeks.