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Aim of this study was to investigate relationships between the red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier) and the entomopathogenic nematode Steinernema carpocapsae (EPN); particularly, the work was focused on the immune response of the insect host in naive larvae and after infection with the EPN. Two main immunological processes have been addressed: the activity and modulation of host prophenoloxidase-phenoloxidase (proPO) system, involved in melanization of not-self and hemocytes recognition processes responsible for not-self encapsulation. Moreover, immune depressive and immune evasive strategies of the parasite have been investigated. Our results suggest that RPW possess an efficient immune system, however in the early phase of infection, S. carpocapsae induces a strong inhibition of the host proPO system. In addition, host cell-mediated mechanisms of encapsulation, are completely avoided by the parasite, the elusive strategies of S. carpocapsae seem to be related to the structure of its body-surface, since induced alterations of the parasite cuticle resulted in the loss of its mimetic properties. S. carpocapsae before the release of its symbiotic bacteria, depress and elude RPW immune defenses, with the aim to arrange a favorable environment for its bacteria responsible of the septicemic death of the insect target.

Rhynchophorus ferrugineus (Olivier 1790) (Coleoptera: Curculionidae) is becoming a serious problem in Mediterranean areas where it is well-adapted, and now is present even in the United States (California). The infestations are primarily in urban areas where chemical control is not advisable and million of Euros are spent to control it. The effects of the entomopathogenic nematode Steinernema carpocapsae (Nematoda: Steinernematidae) on mortality, growth, as well as the immune activity of R. ferrugineus larvae, were investigated. R. ferrugineus mortality exhibited a positive trend with the dosage and duration of exposure to S. carpocapsae. The median lethal dose and median lethal time, important to optimize the treatments, were calculated. S. carpocapsae also had a detrimental effect on R. ferrugineus weight. In vivo and in vitro effects of S. carpocapsae on the phagocytic responses of R. ferrugineus hemocytes also were recorded. S. carpocapsae was not encapsulated by R.ferrugineus hemocytes. After 24 h, the number of hemocytes recorded in treated larvae was reduced. To investigate the defensive abilities of R. ferrugineus humoral and cellular immune systems, specifically against the bacterium Xenorhabdus nematophila (Enterobacteraceae), the minimum inhibitory concentration that inhibits bacterial growth was measured. This is the first time that this technique is applied to entomopathogenic bacteria.

L’adattamento e la conseguente selezione naturale ha prodotto un’infinita varietà di organismi viventi, ciascuno dotato di peculiari caratteristiche, per consentire ad ogni specie di utilizzare l’ambiente in cui vive. I membri che formano le comunità naturali creano tra di essi dei complessi sistemi fatti di rapporti di competizione e di cooperazione. Questi, contribuiscono ad assicurare l’evoluzione delle specie nell’efficace opera di utilizzazione dell’ambiente. Nell’ambito delle relazioni tra individui di specie diverse esistono alcune singolari associazioni che si evolvono insieme, stabilendo un legame intimo e duraturo che comporta un elevato grado di specializzazione da parte dei contraenti. Queste particolari relazioni ecologiche, nelle quali partners diversi concorrono a formare un’unità funzionale di natura permanente o comunque prolungata nel tempo, si dicono simbiosi. In mare le simbiosi sono particolarmente frequenti e spesso hanno aspetti peculiari. Si riportano diversi esempi, quali quelli presenti nella barriera corallina o quella tra un nudibranchio ed alcune alghe unicellulari. Inoltre si evidenzia e si cerca di spiegare le motivazione della quasi assenza degli insetti in mare: forse una mancata simbiosi?

To study the pathogen-host relationship, we used the model of the entomopathogenic bacterium Bacillus thuringiensis (Bt) and Rhynchophorus ferrugineus, a quarantine pest that attacks the palm trees. In particular, we focused on the Bt stress-induced infections. We studied the effect of Bt on larval growth, on hemocytes and on the expression of the heat shock proteins (Hsp70). HSPs are rapidly synthesized in the cells after a stress exposition including pathogens. The Hsp70 was evaluated in the supernatant of the hemocyte lysate (HLS) obtained from larvae fed with Bt. This is the first time that the presence of Hsp70 has been recorded in R. ferrugineus. Bt has negative effects on larval growth, on total hemocytes counts and on the hemocyte type. In addition Hsp70 expression was modulated in the time (3h, 6h, 12h, 24h) in the response to Bt ingestion, highlighting that Bt is a stress factor for the R. ferrugineus. Further investigation is needed to understand the possible correlation between the reduction of hemocytes and Hsp70 modulation.

It is essential to assess the environmental risk that Bt canola resistant to Lepidopteran pests may hold and to study its effect on species assemblages that fulfil a variety of ecosystem functions. Environmental risk assessment can be improved through the use of an ecological model which can be applied to a specific environment, so that local species can be classified functionally and prioritized to identify potential test species. Several other Lepidoptera species are also directly exposed to Bt toxin. In this paper an ecological approach was followed for selection of non-target Lepidoptera species for ecological risk assessment of Bt canola in Sicily, using data collected over a one-year period on Lepidoptera biodiversity on different habitats. Non-target Lepidoptera most likely to be affected were identified and prioritized for future testing and inclusion in risk assessments. Through use of the selection matrix, knowledge gaps were identified for future research and guidance for the design of ecologically realistic experiments.

Increasing body of research deals with the impact of agricultural products marine environment. Indeed it is expected to detect insecticides, including the ones for biocontrol, in nearshore marine waters and thus predicting where, when and with what magnitude these impacts are most likely to occur is imperative if we are effectively interested in understanding the role of biological products as Bacillus thuringiensis (Bt) on marine biodiversity. Bivalves are important elements of marine biodiversity and very often they play a role of ecosystem engineers in that many other species structurally depend on their presence. Oxygen consumption through respiration in these organisms varies with natural physical and chemical factors like temperature and food availability. However, it has been demonstrated that also several contaminants may affect respiration rates in intertidal bivalves like the small Mediterranean Mytilid, Mytilaster minimus. Since the respiration is an important component of energy budget, deviation of its magnitude from common natural patterns may alter the amount of energy available for growth and reproduction. Such a fact, by cascade, may induce alteration of biodiversity. Experiments have been carried out with adult mussels of about 1.4 ± 0.3 cm collected in March 2010 from the coasts around Palermo (Northern Sicily). We measured standardised respiration rates (µmoli h-1 g-1) at ambient temperature (20.0 ± 1.0°C) in mesocosmal tanks both in presence of Bt and without. The significance of the results and the importance of the methods to detect chronic exposure is discussed.

Il batterio Bacillus thuringiensis (Bt) è un patogeno di molti insetti ed è ampiamente impiegato, sottoforma di spore, nella lotta biologica. La forma vegetative (Btv) è considerata quella responsabile della setticemia dell’ospite dopo che le tossine (Cry) rilasciate dal corpo parasporale del Bt in forma sporulata (Bts) hanno perforato il mesenteron del ospite. Tuttavia poco è conosciuto relativamente ai meccanismi di difesa dell’insetto nelle fasi successive alla azione delle tossine Cry. Lo scopo del presente lavoro è quello di studiare gli effetti del Btv sulle larve del fitofago Rhynchophorous ferrugineus Olivier (Coleoptera: Curculionidae) a seguito di iniezioni sub letali di Btv. Questo insetto, noto come il punteruolo rosso delle palme, è stato da noi già preso come modello per studiare l’interazione il Bt e l’ospite ed è stata messa in evidenza la capacità del Bt di invadere, di passare dalla forma sporulata a quella vegetativa e di replicarsi nel curculionide. Dosi sub letali di Btv sono state quindi iniettate nel vaso dorsale di larve di R. ferrugineus dal peso di circa 3 grammi. Dopo 24 – 48 h si è registrata la mancanza di incremento ponderale, in alcuni casi anche una riduzione di peso, delle larve trattate rispetto al controllo. Si sono valutati inoltre gli effetti sugli emociti cimentati con dosi subletali di Btv sia in vivo che in vitro. I risultati mostrano che a seguito dell’iniezione del Btv non solo il numero totale di emociti era drasticamente ridotto ma anche la composizione della popolazione emocitaria era diversa da quella del controllo. In particolare i plasmatociti sembravano essere le cellule maggiormente modulate. Infine l’analisi microscopio elettronico a scansione mostrava inoltre che gli emociti perdevano le loro tipiche morfologie ed emettevano prolungamenti citoplasmatici che li connetteva tra essi.

Heat shock proteins (Hsps) are rapidly synthesized within stressed cells after exposure to an environmental stressor. A variety of environmental stresses, including heat, cold, trace-metal exposure, xenobiotics have been reported to modulate Hsps expression in various organisms. Hsps are grouped into several families based on their protein size. Most organisms have several genes encoding members of this Hsp family. In particularly Hsp70 can be induced quickly under stressful conditions, but return to a normal expression level under non-stressful conditions. Few studies have been done to detect the Hsp70 expression in phytophagous insects towards pathogens. Since a preliminary research disclosed that Bacillus thuringiensis (Bt) negatively interacts with R. ferrugineus circulating hemocytes so that their number was dramatically decreased. In the present research we examine the expression of Hsp70 in hemocytes from R. ferrugineus larvae feed with a commercial product based on Bt. Western blot analyses using monoclonal anti-HSP 70 antibody showed that the expression of Hsp 70 was modulated reaching the highest value, seven times highest to the control, after 3h from the treatment. The Hsp70 values had the same value of the control at 6 hours. Monitoring the Hsp70 for 48 hours we notice a further decrement. This result highlights a stress condition, caused by Bt, as showed also by the reduction of the larval weight. So Hsp70 may be a suitable tool to detect rapidly stress condition induced by potential entomopathogens.

The bacterium Bacillus thuringiensis (Bt) is a pathogen of many insect species and is actively used in biocontrol. The vegetative form as been reported to be involved in insect septicemia process. Bt during the vegetative stage of growth, is known to secern a new family of insecticidal proteins. Moreover recently evidence has been provided, that B. thuringiensis can establish itself in replicative and vegetative form on the leaf surface. Little is known on the interaction of pathogens with the defense responses of phytophagous insects. Insect circulating hemocytes are primarily responsible for the immune defense against parasites and pathogens. We use as model Rhynchophorous ferrugineus Olivier (Coleoptera: Curculionidae) to study the interaction between Bt and insect hemocytes. R. ferrugineus is a key pest of palm trees who are still difficult to combat with both chemical and biological control. Here, we report on the response of larvae of R. ferrugineus circulating hemocytes following Bt vegetative form inoculation. In the hemolymph, plasmatocytes, granulocytes, prohemocytes, oenocytes and spherulocytes were identified. After injection of a sub lethal dose, of Bt, RPW larvae had a decrement in the total number of circulating hemocytes. Particularly there was a reduction in the plasmatocytes which also lost their typical spindle-shape as shown by light and TEM microscopy. However further research are necessary to clarify the role of plasmatocytes in the larvae and their interaction with Bt.

The development of an assay to determine insecticidal properties for either biological and conventional plant protection products plays an important role on the early screening of potential pathogens or derived toxins candidates. The standard methods for the evaluation it has been by bioassay, especially determination of LD50 or LC 50 requiring the use of relatively large numbers of insects and toxin tests. There are several problems connected with these bioassays: availability of insects and in the right life stage, mass producing the candidate species, preparation, reproducibly and costs relative to intensive manpower. These aspects are really important especially when bio-insectides should be tested against new target eg. invasive species in a new country. We present the results of the use of alternative systems in a screening study of Bacillus thuringensis (Bt) strain as potential pathogen for the Red Palm weevil, Rhynchophorous ferrugineus. We propose the use of different techniques such as growth inhibition, effect of pathogen on total number of hemocytes, ratio of selected hemocytes, espression of HSP 70 protein. In particular this protein is involved in the biochemical pathways response in stressed animals. Tests were carried out against the potential less sensitive stage to have a new predictive parameter for screening the reaction of the host to the pathogen. The preliminary results obtained with the proposed tests allows to enlarge the information of the potential pathogenicity of the entomopathogen bacteria and highlight the potential of this set of tests as screening methods to select new potential entomopathogens or bioinsecticides.

In invertebrates, cellular and humoral components are evolved to maintain their homeostais and integrity. Both these components respond to different non-self such as microorganisms, vertebrate erythrocytes and foreign proteins. The aim of this research was to study the possible modulating effects of Bacillus thuringiensis (Bt) in a commercial and vegetative form on the immune response of the echinoderm, sea cucumber Holothuria tubulosa. Bt is one of the most used biopesticides worldwide, covering the 90% of the organic market. Sicily is a region vocated to biological control and agricultural area are often closed to the coastal area. Investigate the effect of biopesticides on other non target organisms is important in a more exhaustive environmental risk assessment. Moreover correlative studies indicate that the immune surveillance is affected by both Bt forms in vivo challenge. In particularly we have evaluated the phagocitosys vs Saccharomyces cerevisiae, hemagglutination using rabbit erythrocytes and coelomocyte cytotoxic activity against rabbit erythrocytes and K562 tumor target cells. These results can contribute to improve the knowledge about the use of Bt like biological insecticidal on non target organisms.

The aim of the present research is to evaluate the impact of agricultural products in the marine environments. Indeed in a country such as Italy and in particularly Sicily where agricultural areas nearshore marine waters, predicting where, when and with what magnitude the impact of xenobiotics derived from agriculture is imperative. In this contest we use as model a biological product (Bacillus thuringiensis) in understanding its potential effects on non target species and on marine biodiversity. Bivalves are important elements of marine biodiversity and very often they play a role of ecosystem engineers in that many other species structurally depend on their presence. Moreover as they consume bacteria, to investigate the effect of the enthomopathogenic bacteria B. thuringiensis on this non target organisms could be useful to implement environmental risk assessment and data on food biosafety. Oxygen consumption through respiration in these organisms varies with natural physical and chemical factors like temperature and food availability. However, it has been demonstrated that also several contaminants may affect respiration rates in intertidal bivalves like the small Mediterranean Mytilid, Mytilaster minimus. Since the respiration is an important component of energy budget, deviation of its magnitude from common natural patterns may alter the amount of energy available for growth and reproduction. Such a fact, by cascade, may induce alteration of biodiversity. Here we focus on effects of two different concentrations of commercial products containing B. thuringiensis (50 and 100 J.II r1) compared with non treated control (i.e., noBt; CTRL) on energy budget of M. minimus. Experiments have been carried out with adult mussels of about 1.4 ± 0.3 cm collected in March 2010 from the coasts around Palermo (Northern Sicily). We measured standardised respiration rates (J.Imoli h-1 g-1) at ambient temperature (20.0 ± 1.00 C) in mesocosmal tanks both in presence of Bt and without. No difference was detected in size of experimental animals. Ingestion rates were significant different among three treatments; respired energy; peaked significantly at 100 J.II r1, and CTRL was different respect to both treatments while assimilated energy was higher in CTRL (87%) than 50 J.II r1 (57%) and 100 J.II r1 (70%). The total amount energy actually absorbed was maximum in CTRL and significantly decreased at 100 and 50 J.II r1. These novel risk assessment strategies may help to more comprehensively