IT201900018065A1 - Insecticidal device - Google Patents

Description

Insecticidal device

Insecticidal device

Technical field of the invention

The present invention relates to an insecticidal device for the control of bloodsucking insects, in particular the device comprises matrices based on biopolymers with "lure and kill" action for indoor and outdoor applications, aimed at controlling the tiger mosquito population (Aedes albopictus ) and other bloodsucking pests (ticks, lice, mites including dermanyssus gallinae, bedbugs, nematodes, annelids, fleas). The biopolymer-based matrix, also the subject of the invention, performs its "lure and kill" action through the biomimetic attraction, the mechanical action, the activity and the self-dissemination of biocides proliferating within it .

The invention finds application in the "house-holding" but also in the veterinary field, animal husbandry, industrial pest control and in large-scale control campaigns, for example in parks, neighborhoods or cities. The invention also relates to ovitraps and other devices and supports that contain or use the matrix according to the invention. In particular, the invention increases the possibility of using such traps, devices and / or supports in the control of pests as it resolves the technical and cost-effectiveness issues described in greater detail in the state of the art.

Known art

The growing incidence of resistance to chemical insecticides, the risks to human health and the environment and the regulations on pesticides have encouraged the search for alternative tools for the control of different species of insect vectors of diseases and in general of pests.

This problem has exploded in conjunction with the worsening of climate change and the globalization of the movement of people and goods that have led to the spread of vectors and pests on a global scale in places where they were not indigenous and in which they have found favorable conditions for their proliferation. . We therefore found ourselves in the position of having to fight new vectors and / or pests with synthetic molecules that are less and less effective if used at concentrations that are safe for humans, animals and the environment. A further detrimental condition is the conveyance of these substances through traditional methods such as the sprinkling / nebulization of aerosols. In fact, the latter, despite being cheap and easy to use, is not very effective in terms of targeting and requires repeated applications over time with increasing concentrations of insecticides, favoring the development of resistance to the active ingredients, increasing the risk of the presence of toxic molecules in the food chain, and the probability of involving insects that are not harmful and essential to the ecosystem such as primary pollinators (eg bees).

Therefore, solutions are sought that have costs comparable to those of the methods used up to now, but which at the same time increase the effectiveness and decrease the environmental impact of pest management actions. To obtain these results, on the one hand, we try to increase the precision of the interventions and on the other hand we use biocides of natural origin, which on the one hand have a lower environmental impact and are less prone to develop resistance phenomena on the part of insects, but on the other hand they have higher costs and greater difficulties of application.

An exemplary case is that of the control of the tiger mosquito (Aedes albopictus), vector of the arboviruses Zika, Dengue, Chikungunya and Yellow Fever. The tiger mosquito has adapted to live in both indoor and outdoor anthropized environments, it has its peaks of activity during the day and at night it prefers to take refuge in humid and difficult to access ravines that it often also uses as oviposition points. It is evident that - both for the impossibility of spraying insecticides in inhabited centers during the day and for the difficulty of targeting during periods of inactivity - specific control strategies and methods are needed and that possibly also involve biocides since it has been resistance to various molecules found (Pyrethroid resistance in Aedes aegypti and Aedes albopictus: Important mosquito vectors of human diseases, Smith LB, Pesticide biochemistry and physiology, 2016 Oct 133: 1-12).

In the specific case of the tiger mosquito, among the possible alternatives there are control through ovitraps (with or without insecticide) and biological control through bioinsecticides such as microbial larvicides (eg Bacillus thuringiensis var. Israelensis) or symbionts (eg Wolbachia pipientis) , natural essential oils or through entomopathogenic fungi such as Beauveria bassiana (BB) and Metarhizium anisopliae.

An ovitrapper is a device used in the past almost exclusively for the collection of eggs for scientific monitoring purposes, then subsequently for the capture of pregnant females. It works based on the habit of female tiger mosquitoes (in natural habitat) to lay eggs (The biology of Aedes albopictus, W. A. Hawley, Journal of the American Mosquito Control Association. Supplement, 1988, vol 42-40):

● on vertical or sloping surfaces, placed in hidden and shaded areas,

● in humid areas (or where they perceive the presence or possible presence of water) and not directly in water,

● in sites free of chemical substances that discourage deposition or in general unpleasant for the insect,

● in environmental conditions of low salinity and pH tending to neutral.

In nature these places are made up of rocky ravines, hollow plants, muddy areas on river banks, etc. In populated areas, similar conditions are found in hidden places such as pots and saucers, waste, abandoned tires, gutters, etc. The temperatures at which hatching takes place range from 20 to 40 ° C.

Therefore, to provide this attractive habitat, a trap in the simplest form consists of a container made of polymeric material filled with water with a vertically placed masonite rod inside. The masonite, when moistened, attracts the tiger mosquito to lay on it. The chopsticks are then removed and the eggs collected. The transition from a monitoring tool to a control device led first to add synthetic insecticides to the water and then to new types of traps, considerably more complex, which avoid the use of insecticides but provide elements for the active capture of adults (eg . fans to suck them) or carbon dioxide cylinders and / or light signals to attract. This type of traps is intended for domestic use due to the need for electricity and the high costs. In attempts to use ovitraps in large-scale control campaigns (therefore with large numbers of traps to be placed on very large areas), the reference trap is the one used for monitoring with the addition of antilarval insecticides and any attractive substances.

These traps, when full of water, even if without the deposition substrate, act as deposition sites also for other species that spawn directly in water (eg. Anopheles, Culex, Aedes) therefore they are not specific for a single species of mosquito.

The problems related to this last class of traps (defined as passive "lure and kill" traps as they do not use elements that do work to attract or capture, such as fans) are:

a) need for constant topping up of evaporated water and insecticide so that they remain functional,

b) continuous monitoring and therefore costs related to personnel, c) use of chemical insecticides with consequent environmental and interaction risk for animals and humans and non-target insects, d) need to recover traps when not active to prevent them from becoming new outbreaks of deposition if filled by rain (and therefore additional costs for recovery and eventual disposal),

e) low effectiveness due to competition with natural deposition sites in their vicinity

The known solutions proposed go in the direction of creating biodegradable traps to deal with problem d) but in any case based on the use of liquid water and insecticide (eg. Greenlid Biotrap).

Another solution is to create large standard traps to increase the duration. In addition to management problems, increasing the duration up to 10-15 days by using traps containing free water allows the larvae to reach the adult stage.

It would therefore be necessary to add larvicidal products to the traps.

The effect of the laying substrate on the quantity of eggs laid in competition with natural sites is not evaluated and neither is the possibility of using substrates that retain water or absorb it from the environment (hydrophilic or hygrophilic) for periods of time compatible with the degradation of the trap or that imitate the natural conditions (also due to the lack of quantitative information regarding the characteristic parameters of the substrates). The role of the substrate is significant as it influences the number of eggs laid (Evaluation of attractants and egg-laying substrate preference for oviposition by Aedes albopictus (Diptera: Culicidae), U. Thavara, Journal of Vector Ecology, 2004, pp 66-72) . The standard substrate is a 20 × 2 cm masonite stick but for example B.C. Zeichner et al. they used high-weight tissue paper soaked in an insecticide solution. Other examples of substrates are found in US patent 9237741 in which a hydrogel based on polyacrylamide (PAM) (therefore synthetic and not biodegradable) is used as a deposition substrate to be inserted in a polyethylene container but also in US patent document 20130303574 in which a gel based on silica and talc is proposed which favors the transmission by contact on the insect of an insect growth regulator or other synthetic insecticides.

In the documents mentioned above the trap:

- attracts pregnant mosquitoes thanks to the presence of attractive substances and water (both in a liquid state)

- kills the adult female by contact with a synthetic insecticide

- the deposition substrate, made of synthetic hydrogel based on polyacrylamide (PAM) or silica, is not biodegradable and is not biocompatible

- the substrate does not foresee and cannot act as a substrate for the growth of any biocide as it is not biocompatible.

Ultimately, there are no studies that quantitatively characterize the factors that influence the choice of the deposition substrate (e.g. water content of the substrate, viscosity, composition, etc.), but operating conditions such as those of wet masonite can be characterized. a free water content by weight between 30-50% calculated by differential scanning calorimetry (as described below), neutral pH and salinity lower than 3% (pH and salinity evaluated under standard conditions and depending on the absorbed solution).

In general, the possibility of hosting biocides replacing synthetic insecticides in the same deposition substrate has not been evaluated, an option that can only be achieved if there is biocompatibility between the material that constitutes the substrate and the biocide, a condition that is unlikely with synthetic polymers, and whether the substrate creates a suitable microenvironment for growth.

Take for example Beauveria bassiana (BB). This entomopathogenic fungus acts as a parasite of some types of insects, infecting them by contact (transmission of "lime" without the need for ingestion but through destruction of the cuticle by some enzymes and release of the toxin beauvericin), colonizing and killing them in a sufficiently long period of time long to allow the process of self-dissemination, that is the dispersion of the fungus by the parasite itself in other places infested or that will be infested by the parasite. Furthermore, the dead insect itself becomes a source of BB spores.

BB has assumed a key role in the management of numerous agricultural, veterinary and forest pests but also for the control of mosquito populations. In fact, it is effective on C. tarsalis, C. pipiens, A. aegypti, A. sierrensis, A. nigromaculis, and A. Albimanus (Field and Laboratory Studies on the Pathogenicity of the Fungus Beauveria bassiana to Three Genera of Mosquitoes, Truman B . Clark et al. Journal of invertebrate Pathology, 1968, 11, 1-7), but also against A. albopictus (Characterization of Tolypocladium cylindrosporum-Hypocreales: Ophiocordy cipitaceae - and Its Impact Against Aedes aegypti and Aedes albopictus Eggs at Low Temperature, American Mosquito Control Association, Journal of the American Mosquito Control Association, 2017 Sept. 33 (3): 184-192).

BB can be mass produced. Mass production usually involves using a mixture of cereals and water in a 1: 1 ratio to which a suspension of BB spores is added. The ideal incubation conditions are 25 ° C and pH 4-8. It has been verified how the water content affects the growth of BB and in particular how the number of conidia produced with the same substrate grows with the increase in the amount of water (Effect of Growing Media and Water Volume on Conidial Production of Beauveria bassiana and Metarhizium anisopliae, M. El Damir, Journal of Biological Sciences, 2006, 6 (2) -269-274).

Beauveria bassiana is typically administered in one or more applications of conidia that are released into the air in dry or liquid formulations but also in aqueous or oily solutions, granules, pellets or floating formulations that exploit the possibility of the spores to multiply in an aquatic environment. The use of BB in biological control does not present risks to human or animal health and its effectiveness against mosquitoes has been verified even when applied on substrates. For example, the spores of BB have been applied by spraying on earth, concrete and wood (Storage and persistence of a candidate fungal biopesticide for use against adult malaria vectors, S. Blanford, Malaria Journal, 2012, 11: 354) and mosquitoes ( in this case Anopheles stephensi and Anopheles gambiae) were exposed for 1h to the treated substrate and then removed. After removal, survival was observed over the next 14 days and survival showed a strong dependence on the BB application substrate (confirming the link between application substrate and BB efficacy). Effectiveness and residual effect were also verified on plywood, bricks, plastic and glass surfaces (Persistence and efficacy of a Beauveria bassiana biopesticide against the housefly, Musca domestica, on typical structural substrates of poultry houses, Naworaj Acharya, Biocontrol Science and Technology , 2015, 697-715).

The use of BB is however severely limited by the persistence times. In fact, a strong dependence of persistence on environmental conditions and on the substrate of application has emerged.

In particular, BB, if applied on earth, has a persistence comparable to that of a common pesticide (up to 4-5 months) while if applied on wood or concrete its persistence is reduced (from 1 week to 2-3 months). Differently if used in operating conditions (exposed to heat, UV radiation and drying of the substrate), the persistence drops to days.

In general, it has been shown that the encapsulation of the conidia of mushrooms within polymeric matrices preserves their vitality and persistence. Procedures of this type are contained in the French Pat patent documents. No. 2,501,229 U.S. Pat. No. 4,647,537 U.S. Pat. No. 4,724,147 U.S. Pat. No. 4,668,512. The inventions described relate to the encapsulation of pathogens within matrices which involve gelling with the use of metal cations and the formation of a silica or silico-aluminate-based gel (Jung et al.) Or formulations based on alginate but in pellets or microspheres, therefore devoid of any attractive functionality for the insect (Shigemitsu, Marois et al. Lewis et al.).

The characteristics of resistance and in particular of tolerance to thermal stresses are also strongly influenced by the composition of the growth medium of BB. For example, if carbohydrates are added to the preparation used for growth, BB is able to withstand temperatures of about 50 ° C (Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana, S. ‐ H . Ying, Applied microbiology, Sept. 2006, Vol. 43 pp 331-335).

US patent 5141744 is moving towards encapsulating the spores of BB and other biocides, relating to the preparation of an insecticidal composition in the form of a hydrated macrogel that contains at least one species of entomopathogen and a compound capable of retaining hydration with the function of acting as a reserve of water for the entomopathogen itself. The purpose of the macrogel is on the one hand to maintain the vitality of the pathogenic organism by providing a useful means for positioning it in insect infestation sites, and on the other hand to act as a bait for insects that are infected upon ingestion ("continuous insect consumable matrix ").

For some insects, food baits are a valuable tool, but not for the vectors of diseases such as mosquitoes, ticks, lice, mites, etc. as bloodsucking. Therefore the attraction with bait (as foreseen by the invention in the aforementioned document) is not effective either in terms of attractiveness, since it develops exclusively food-like attractiveness, nor in terms of infection since the biocide can only act if ingested. Furthermore, the gels described in US 5141744 are mixtures of matrices of natural origin such as oligopolysaccharides to which synthetic gels based on acrylic polymers, polyacrylamides and polyurethanes, which are not biodegradable, are added. Also said gels:

a) contain chemical crosslinkers which, in the case of the mosquito, can act as a deterrent for the deposition in addition to being toxic for the bioicides used,

b) have no control over the amount of free water necessary for the survival and proliferation of the biocide and the lure and kill action, for example against mosquitoes (free water in the optimal configuration is between 70-80% by weight),

c) they do not provide any indication on the rheological or yield stress properties that guarantee the possibility of use on vertical walls.

The material proposed in US 5141744 acts exclusively as a protection to the pathogen and, as reported in the examples, does not show any growth of the biocide charge in the matrix rather, on the contrary, a reduction already after one week. Furthermore, the absence of nourishment does not favor the proliferation and survival of the pathogen for long periods.

There is also the disadvantage of dispersing a non-biodegradable synthetic polymer into the environment which, moreover, could also be ingested by non-target animals, children, insects, incurring limitations of use and therefore contradicting the need for innovation previously reported.

Similarly, US patent 5273749 describes a process for the coating of microbial pesticides but such as to be applied on leaves against plant weeds and subsequently ground and processed to be applied by spraying on plants or seeds. The invention has no lure and kill characteristics nor has it optimized characteristics to allow a type of biomimetic attraction. In fact, the proposed formulation is designed to be sprayed on already infested leaves or soil or seeds and roots or for preventive purposes as would be done with any other insecticide. No characteristic of the gel that simulates an environment or condition that is naturally attractive to pests is sought or specified. The only role of the material is to provide a protective environment for the bioactive substance that must act in the infested environment, without any entrapment functionality or the possibility of mechanical killing by suffocation of larvae or eggs.

Therefore, the problems posed by the known art remain unresolved to date.

SUMMARY OF THE INVENTION

The object of the present invention is an insecticidal device consisting of a matrix with "lure and kill" operation, intended for the control in indoor and outdoor conditions of the population of bloodsucking pests (including also possible vectors of diseases) such as mosquitoes including the tiger mosquito, bedbugs, ticks, lice, mites including Dermanyssus gallinae, nematodes, annelids, papadaci, fleas etc.

The matrix object of the invention simultaneously performs the "lure" functionality by exploiting the principle of biomimetic attraction and the "kill" functionality through mechanical action or with the aid of biocides, as well as performing the functionality of "survival and growth of the biocide" (i.e. to keep the biocide used for the target insect vital and effective and / or to proliferate) by exploiting the biocompatibility of the materials and the creation of a suitable microenvironment.

The invention is functional only when it has the characteristics suitable to simultaneously satisfy each functionality.

The matrix is prepared in the form of a gel in spreadable and / or pourable and / or moldable hydrated form or in dehydrated form. The gel is based on totally biodegradable, biocompatible, hydrophilic and hygrophilic biopolymers (i.e. able to absorb humidity and retain large quantities of aqueous solutions for prolonged times). By varying the composition and the concentration of polymer, based on the type of polymer used, the parameters (e.g. pH, salinity, free water content, yield stress, etc.) that determine the properties necessary for operation, or for simultaneous satisfaction, vary. of all features.

In practice, the matrix of the invention possesses the characteristics suitable for attracting a hematophagous insect by mimicking the characteristics of a natural habitat ideal for it for carrying out specific vital functions (including reproduction). For example, in the preferred composition, which has the tiger mosquito as its target insect, the matrix must mimic an egg-laying environment and for this reason it has a pH in the range 4-8, salinity in the range 0-3% (intended as a concentration of salts present in the solution and measured by conductivity measurements under standard conditions), yield stress threshold value of not less than 50Pa necessary for the gel to be spreadable and not pour on a vertical wall (measured with a rotational rheometer under standard conditions), a content of free water (i.e. not chemically bonded to the substrate) in the range 70-80% by weight (measured under standard conditions) and a viscosity of not less than 2 Pas.

At the same time, the matrix is able to kill the insect and / or its eggs and / or larvae through the mechanical action of the matrix itself and / or thanks to the presence of a biocide. The mechanical action is carried out with the entrapment and consequent suffocation of the eggs and larvae within the polymer matrix, an example is shown in Figure 10.

If a natural biocide is used (eg entomopathogenic bacteria or fungi, etc.), it can survive and proliferate within the matrix ("survival and growth" functionality) up to lethal concentrations for the target insect and / or for the its eggs and / or larvae.

For example, in the preferred composition intended for the tiger mosquito, the conditions necessary for the "lure" functionality are suitable for the proliferation of the biocide Beauveria bassiana up to conidic concentrations of 10 <6> -10 <7> conidia / mg of matrix, required quantity to the "kill" functionality.

A further object of the invention is a matrix in hydrated, dehydratable and rehydratable form, which can be applied on a support, preferably biodegradable, able to be possibly inserted inside traps or other devices or containers functional to the creation of the attractive habitat or to the delivery of the matrix (such as in the case of containers in mosquito traps).

In the dehydrated form the matrix can be pre-shaped to be inserted into / on a support. The matrix will be pre- or subsequently rehydrated with a predetermined quantity of water until obtaining the same characteristics as the original hydrated matrix.

Still another object of the invention are ovitraps and other devices and supports that contain or use the matrix according to the invention.

The matrix and the devices of the invention find application not only in the field of "house-holding", but also in the veterinary field, animal husbandry, industrial pest control and in large-scale control campaigns, for example in neighborhoods, parks or entire cities.

Further objects, purposes and advantages will become evident from the detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Cross section of a trap containing the matrix.

Figure 2. Viscosity values as the weight concentration of 2-hydroxyethylcellulose (2-HEC) varies.

Figure 3. The curve shows the values of free water in the matrix as the% wt (percentage by weight) of 2-hydroxyethyl cellulose (2-HEC) varies.

Figure 4. Yield stress (Pa) values for different 2-HEC concentrations.

Figure 5. Loss of water over time for a matrix made as in example 1 and tested in a climatic chamber inside a trap like the one in figure 1.

Figure 6. T / C values as a function of the percentage by weight of 2-hydroxyethyl cellulose (% wt 2-HEC). T is the number of eggs deposited on the matrix compared to that deposited on control C. The curve shows the values of free water in the matrix as the% wt of HEC varies.

Figure 7. Conidic composition-growth relationship (2-HEC with and without peptone). Evaluation of growth at the same concentration by weight of 2-HEC in matrices with and without peptone compared with two suspensions in distilled water with Beauveria bassiana (conidia and mycelium) respectively with and without the addition of peptone or other nutrients called respectively G and H.

Figure 8. Comparative lethality test between matrices and suspensions previously subjected to growth test. The reference control against which mortality was assessed is distilled water (not shown in the figure).

Figure 9. Comparative lethality test between matrices without Beauveria bassiana with weight concentrations of 2-HEC between 2-30% prepared as in example 1. G and H suspensions in distilled water of Beauveria bassiana (conidia and mycelium) respectively with and without the addition of peptone. K: distilled water with the addition of 1% by weight of peptone. The reference control against which mortality was assessed is distilled water. Yield stress and threshold yield stress curves are also shown.

Figure 10. Example of the mechanical action of the matrix and the effect of BB. A) Stereo microscope image of a larva trapped in the matrix. B) electron microscope image of an egg and the structures of the fungus grown on it.

Figure 11. The curves show the values over time of the number of conidia of Beauveria bassiana within the matrix according to the composition shown in example 1 (A) and in example 2 (A freeze-dried). The stored A curve shows the values of the number of conidia over time present within the lyophilized matrix and stored in a closed container and stored under standard conditions.

Figure 12. Deterrent effect of the crosslinking process on the matrix oviposition made according to example 3.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is a matrix with activity and with a "lure and kill" function and devices with an insecticidal function that contain said matrix, intended for the control in indoor and outdoor conditions of the population of bloodsucking pests (including also possible vectors of diseases) such as mosquitoes including the tiger mosquito, bedbugs, ticks, lice, fleas, mites including Dermanyssus gallinae, nematodes, annelids, papadaci, etc.

The matrix object of the invention simultaneously performs all the following functions. The "lure" functionality, carried out by exploiting the principle of biomimetic attraction; the functionality of "survival and growth of the biocide" (that is to maintain vital and effective and / or to proliferate the biocide used for the target insect) which is carried out by exploiting the biocompatibility of the materials and the creation of an ideal microenvironment and functionality "Kill" which is carried out by mechanical action or with the aid of biocides.

The invention is functional only when it has the characteristics suitable to simultaneously satisfy each functionality.

The matrix is prepared in the form of a gel in a spreadable and / or pourable and / or moldable hydrated form or prepared in a dehydrated rehydration form. The gel is based on biodegradable, biocompatible, hydrophilic and hygrophilic biopolymers (capable of absorbing humidity and retaining large quantities of aqueous solutions for prolonged times).

By varying the composition and concentration of polymer, based on the type of polymer used, the values of those parameters (e.g. pH, salinity, free water content, yield stress, etc.) that determine the characteristics necessary for operation, i.e. to the simultaneous satisfaction of all the functions specified above.

In practice, the matrix of the invention possesses biomimetic characteristics, that is, the characteristics suitable for attracting a hematophagous insect by mimicking a natural habitat ideal for it for carrying out specific vital functions (including reproduction).

At the same time the matrix is able to kill the insect and / or its eggs and / or larvae through the mechanical action of the matrix itself, an activity linked to the entrapment and consequent suffocation of the eggs and larvae to the inside the material (an example is shown in figure 10) and / or thanks to the presence of a biocide. In particular, if a natural biocide is used (eg entomopathogenic bacteria or fungi, etc.), the latter can survive and proliferate within the matrix ("survival and growth" functionality) up to lethal concentrations for the target insect, for its eggs and / or larvae.

A further object of the invention is a matrix in hydrated form that can be applied on a support, preferably biodegradable, able to be possibly inserted inside traps or other devices or containers functional to the creation of the attractive habitat or to the delivery of the matrix ( for example as in the case of containers in mosquito traps).

In the dehydrated form the matrix can be pre-shaped to be inserted into / on a support. The matrix will then be rehydrated with a predetermined amount of water until the same characteristics of the desired hydrated matrix are obtained.

Still another object of the invention are ovitraps and other devices and supports that contain or use the matrix according to the invention.

These inventions find application in the "house-holding" but also in the veterinary field, in animal husbandry, in industrial pest control and in large-scale control campaigns, for example in neighborhoods, parks or entire cities.

For the purposes of the description and within the scope of the present invention, the following definitions are given.

Functionality is defined as a specific task that the invention must perform.

"Lure" functionality is defined as the ability to attract the target insect. "Kill" functionality is defined as the ability to kill the target insect and / or its larvae and / or its eggs.

"Survival and growth" functionality is defined as the ability of the invention matrix to maintain vital and effective and / or to proliferate the active substance (biocide) used against the target insect. The term "biomimetic attraction or biomimetic lure" means a way of carrying out the "lure" functionality not with traditional attractive substances, such as sexual aggregation pheromones and / or food attractants and / or other synthetic aromas with behavioral influence on 'insect, but providing the insect with a habitat with ideal conditions (mimicking those present in nature or using more attractive ones) for carrying out specific vital functions (including reproduction or development of offspring) typical of the target insect .

The term "kill by mechanical action" means a method of carrying out the "kill" function attributable to the action of the matrix alone without biocides or other active substances carried out through the entrapment in the matrix of eggs that are unable to hatching or suffocation of any larvae (Figure 10). To carry out this action, the matrix does not need a biocide inside but it is a capacity which, in the preferred formulation, is inherent in the material itself and linked to the viscosity of the material.

With the words "kill (kill) through the action of biocides and / or active substances present in the matrix" we mean a way of carrying out the "kill" function attributable to the action of infection by biocides and / or other active substances present in the matrix.

By "biocidal product" we mean any substance or mixture (or generated from substances or mixtures) consisting of, containing and / or capable of generating one or more active ingredients, in order to destroy, eliminate and render harmless, prevent action or exercise other control effect on the target harmful organisms of the invention, by any means other than mere physical or mechanical action.

The biocides of natural origin that can be used in the invention include Gram-negative and Gram-positive bacteria, actinomycetes, fungi, protozoan yeasts, algae and their spores. Some examples are Bacillus subtilis subsp. Krictiensis, Pseudomonas pyrocinia, Pseudomonas fluorescence, Gliocladium wirens, Trichoderma reesei, Trichoderma harzianum, Trichoderma hamatum, Trichoderma viride and Streptomyces cacaoi subspecies asoensis; or microorganisms such as Bacillus thuringiensis. In particular, an example of a preferred biocidal group that can be used within the present invention consists of the entomopathogenic fungi belonging to four main groups: Oomycota (Lagenidium and Leptolegnia), Ascomycota (including Aspergillus, Beauveria bassiana, Metarhizium, Paecilomyces) and Zygomycota ( including for example Conidiobolus, Entomophaga, Entomophthora, Erynia, Furia, Massospora, Neozygites, Pandora, Zoophthora). These substances are added to the starting solution to be gelled in variable quantities depending on the type of biocide and subsequently proliferate in the matrix object of the invention. For example in the case of Beauveria bassiana used against the tiger mosquito the starting concentration is 10 <3> -10 <4> conidia / mg of matrix with proliferation up to 10 <6> -10 <7> conidia / mg of matrix .

By "bloodsucking insects" we mean that class of insects which have as a food behavior that of feeding on the blood of a vertebrate. In the present invention we mean by bloodsucking arthropods those systematic groups of organisms belonging to arthropods which have as their alimentary behavior that of feeding on the blood of a vertebrate: by way of explanation but not exhaustive, different species belonging to the orders Ixodida (hard and soft ticks), Mesostigmata (parasitic mites), Anoplura (lice), Siphonaptera (fleas), Hemiptera Eteroptera (bedbugs), Diptera (mosquitoes, sand flies, horseflies, (Tabanidae), horse flies (Stomoxys calcitrans), etc.). In particular, as regards mosquitoes, reference is made to the genus Aedes, Culex, Anopheles.

The wording "biocompatible and biodegradable biomaterials" means natural hydrogels and among the most commonly used we can mention those belonging to the classes of: alginates, cellulose including for example hydroxyethyl cellulose, carboxymethylcellulose, pectins, polysaccharides, chitosans and agar. The biomaterials are added individually or in a mixture to the solution containing the biocide in concentrations that vary according to the biopolymer used. For example, the concentrations that allow the operation against the tiger mosquito using the biopolymer 2-hydroxyethylcellulose and Beauveria bassiana as biocide, are between 10-20% by weight. While using, for example, carboxymethylecellulose, the operating conditions are satisfied for concentrations between 2-10%.

Within the invention, a nutrient substance is intended as any element (or mixture of elements) which, added to the matrix, is useful for the life and metabolism of the living organisms present within it. Among the nutrients are considered carbohydrates, lipids and proteins, long chains of amino acids, minerals and vitamins in their simplest or complex forms and all their derivatives. For example, in the formulation used against the tiger mosquito, in the presence of Beauveria bassiana, 1% by weight of peptone (a water-soluble powder mixture of peptides and lipids) is added as a nutrient.

In the invention, a gel is intended as a biopolymer-based preparation consisting of a liquid dispersed and incorporated in the solid phase, which includes or does not include biocides or nutrients that can be pourable, a spreadable or moldable paste, etc. obtained by adding a biopolymer to an aqueous solution or following rehydration of a previously dried matrix. In general, to ensure the operation of the invention, the parameters that characterize the gel, common to all functions (obtained experimentally or in accordance with the literature reported in the state of the art), are the following (measured under standard conditions): salinity not higher than 3%, pH 4-8, viscosity between 10 <-3> -10 <4> Pas and / or storage modulus and / or loss modulus values between 0-2MPa, yield stress values between 0 -2000 Pa, in addition to a free water content above zero and preferably between 5-98% by weight (measured by differential scanning calorimetry). The biomaterials that make up the gel are added individually or in a mixture to the liquid in concentrations that vary according to the biopolymer used but such as to ensure that the gel respects the ranges of parameters reported.

Within the invention, a humectant substance is intended as any element (or mixture of elements) with hygroscopic characteristics of natural origin which, added to the matrix, is useful for reducing dehydration and in general for maintaining the characteristics necessary for the performance of the previous functions. defined. These include (as they are non-toxic and biocompatible as they are already used in the food industry) glycerin, propylene glycol, glyceryl triacetate sorbitol, xylitol and maltitol, polydextrose or natural extracts such as quillaia, compounds such as acid lactic or urea.

In the description of the invention, "quantity of free water" means that quantity of water / aqueous solution contained in the gel and not chemically linked to the biomaterial. It is expressed as a percentage of the weight of the gel and measured by differential scanning calorimetry with the method reported below.

In the description of the invention, salinity is understood to mean the concentration of salts dissolved in the gel which has been measured by electrical conductivity under standard conditions and is expressed as a percentage to be understood as grams of solute for every hundred grams of solution (mass fraction).

Therefore, for the purposes of the present invention by bio-matrix with lure and kill action through biomimetic attraction and mechanical action and of biocides proliferating inside it (called matrix for simplicity) we mean in the most general sense a preparation in the form of a gel which, possessing technical characteristics within the ranges shown, it allows to simultaneously perform the “lure”, “kill” and “survival and growth” functions defined above in the defined methods.

Method for preparing the matrix.

The matrix according to the invention is prepared with the following basic steps:

i.) In a temperature range of 15-60 ° C prepare an aqueous solution and add the polymer until gelation, continuing to mix at a constant temperature.

The aqueous solution can be previously prepared so as to contain a biocide and at least one nutrient for said biocide before adding the polymer and / or a wetting agent. For example, in the case of the matrix intended for the control of the tiger mosquito, conidia and mycelium of Beauveria bassiana are added to the starting aqueous solution until a turbidity equal to 2 McFarland is obtained together with peptone (1% by weight) and finally the polymer ( e.g. 2-HEC, 16% by weight), mixing until gelation.

ii.) As an optional additional step: pour into a mold and dry. Preferably, if the matrix contains biocides, eliminate water, for example freeze-dry by freezedrying.

As indicated above, the matrix can be prepared in dehydrated form, for example with the freezedrying technique or other similar technique, known per se. In this embodiment, a matrix, for example based on 2-hydroxyethylcellulose which includes and allows the entomopathogenic fungus Beauveria bassiana to proliferate, is first prepared in the form of a gel, then it is poured or injected into a mold and subsequently subjected to the method freezedrying or dehydration by sublimation at low temperature and low pressure. With this method, the entomopathogen remains vital but quiescent within the matrix and resumes its activity when the matrix is rehydrated. The matrix is easier to store and easy to use as it is already shaped and only needs to be wet to perform its function.

As previously reported, each function has parameters with defined operating ranges. The invention is functional when it satisfies the common conditions of operation for the different functions.

To ensure the functionality of "biomimetic lure" the matrix must have the following characteristics:

● it contains a quantity of free water higher than 0 and preferably between 5-98% by weight, in the case of the tiger mosquito in the preferred composition between 70-80% to be higher than the standard control,

● does not contain in the formulation of substances that are repellent, for example for mosquitoes substances such as chlorine and in general cations (eg copper), aromas (eg lemongrass and the like), chemical insecticides, acids, bases etc.,

● is processable in the form of a gel capable of covering a surface uniformly. In the case of the formulation against the tiger mosquito, a support must be completely covered with a gel thickness between 2-10 mm,

● possesses, at the useful polymer concentrations suitable for the other functions, Yield stress values such as to be able to be applied even on inclined walls. For example, in the case of the tiger mosquito, in order for a 5mm layer not to leak if placed on a vertical wall, the matrix must have yield stress values greater than or equal to 50 Pa (measured under standard conditions).

● pH between 4 and 8,

● maximum salinity of 3%,

● maintains at least 5% free water by the fifteenth day of exposure under service conditions. For example, the preparation for the control of the tiger mosquito, in order to remain attractive, must maintain at least 35% of free water on the fifteenth day of stay in the typical environmental conditions of the tiger mosquito deposition sites (18-30 ° C relative humidity above 70%),

● contains biopolymers in concentrations such as to guarantee the previous characteristics,

● contains wetting agents in concentrations such as to guarantee the previous characteristics. In the case of preparations for the tiger mosquito in concentrations ranging from 0-10% by weight.

To ensure the performance of the "biocide survival and growth" functionality, the matrix must have the following characteristics, suitable for maintaining a habitat favorable to their survival, proliferation and infectious activity:

● contains at least 5% by weight of free water, a quantity that is also valid in the specific case of the preparation in which Beauveria bassiana is used,

● pH between 4 and 8,

● maximum salinity of 3%,

● it is prepared with processes that are not directly harmful to the vitality of the biocide or alter the conditions of the matrix necessary for the "survival and growth" functionality. For example, in the formulations in which the use of entomopathogenic fungi is foreseen, a temperature higher than 50 ° C cannot be used or possibly sterilization methods of the BB-containing matrix that provide UV or extra heat cannot be subjected to dehydration obtained with high temperature.

● it is composed of biopolymers and nutrients and / or wetting agents and / or other substances that are biocompatible, ie non-toxic towards the biocide, and in concentrations such as to respect the listed characteristics.

The "kill" functionality (both mechanical and linked to the biocide) is guaranteed by the following characteristics of the matrix:

● performs an action of drying and / or trapping on eggs and / or larvae and / or adults (mechanical action) regardless of the presence of the biocide which is linked to the viscosity of the matrix. In the case of the preparation intended for the control of the tiger mosquito the viscosity value is not less than 2 Pas,

● allows the proliferation of the biocide up to lethal concentrations for the target insect, for example in the case of Beauveria bassiana it is necessary to reach concentrations of 10 <6> -10 <7> conidia / mg of matrix to be maintained for at least 10 days so that is effective against tiger mosquito eggs and larvae,

● allows the self-dissemination of the biocide if present, or the possibility of the matrix containing the biocide (or the biocide itself) to be transported to other places by the insects themselves that come into contact with the matrix. This occurs for viscosity values below 10 <2> Pas (measured under standard conditions) and quantities of free water compatible with the “biocide survival and growth” functionality.

Furthermore, the matrix of the invention can be used inside devices or on supports, remedying the problems of the lure and kill type devices for the tiger mosquito (ovitraps) and other bloodsucking insects, if, in addition to satisfying the reported functionalities, has the following characteristics:

● it is made with completely biodegradable polymers, ● it has a formulation with elements with a low risk profile towards humans and animals and the environment,

● it has a durability, i.e. maintenance of the parameters necessary for functionality in a suitable range of values, of at least fifteen days in the classic conditions of the places where the tiger mosquito is deposited (temperatures between 18-30 ° C and humidity not less than 70%) .

The advantages associated with the matrix object of the present invention will be summarized below:

● it is multifunctional, that is, it performs several functions at the same time: "biomimetic lure", "survival and growth of the biocide" and "kill", not limited to being only an attractive substrate or only for growth or only lethal,

● targets a class of insects that cannot be controlled with baits, on the contrary it can use biocides (such as entomopathogenic fungi including Beauveria bassiana) that do not need to be ingested to act,

● does not use synthetic pesticides but can use natural biocides.

This ensures that the insect does not develop resistance, a low environmental impact and a potential absence of risk for humans and animals since no chemicals that can be toxic are used and consequently a wider use scenario,

● it is selective, as it allows to harm a specific harmful insect but not to other organisms that will not be attracted by the conditions mimicked by the matrix;

● thanks to the hygrophilic and hydrophilic materials of which the gel is composed and to its complete biodegradability, it can be used for prolonged times without maintenance, reducing service costs during disinfestations and eliminating disposal costs,

● it is easy to prepare, as all preparations are at room temperature,

● it is easy to use and apply as it can be spread by the user or from a coating line or dehydrated and rehydrated directly on the support until the initial conditions are obtained,

● manages to grow and develop the bioinsecticide up to concentrations such as to be lethal for the target species.

According to a preferred embodiment, the matrix is intended for the control of the tiger mosquito (Aedes albopictus) and is a preparation based on 2-hydroxyethylcellulose (2-HEC) which includes and allows the entomopathogenic fungus Beauveria bassiana to proliferate inside it. It is made in the form of a gel with characteristics such as to simultaneously perform the functions of "biomimetic lure" being spreadable and acts both as an egg-laying substrate, attracting the tiger mosquito to spawn, and as a lethal matrix through the mechanical action of the polymer and of the Beauveria bassiana proliferating in the matrix. In addition, the mosquito, contaminated by the entomopathogenic fungus, will ensure self-dissemination in other spawning sites. The use of the matrix inside a trap is shown in Figure 1 which shows a cross-section of a set up for using a trap with the matrix inside which is placed in direct contact with the vertical walls.

The invention remedies the dangerous and ineffective use of synthetic insecticides (especially due to the resistance developed by insects and the difficulties of targeting some species) using a "smart", inexpensive, extremely selective, eco-sustainable approach and with substances with low risk profile that can be used without major restrictions. In particular, the invention increases the possibility of using such traps, devices and / or supports in the control of pests in indoor and outdoor contexts and also in large-scale campaigns as it resolves some technical problems that compromised the cost-effectiveness of the systems listed in the state of the art.

The following examples are intended to illustrate the present invention, without limiting its scope of protection. It will be apparent to those skilled in the art that some modifications can be made to the present invention without departing from the spirit or scope of the invention as set forth herein.

Example 1: preparation of a matrix based on 2-hydroxyethylcellulose (2-HEC) peptone and Beauveria bassiana (BB), intended for the control of the tiger mosquito (Aedes albopictus).

According to a preferred embodiment, the matrix is intended for the control of the tiger mosquito (Aedes albopictus) and has the following composition: distilled water, 20-300 g / liter of 2-hydroxyethylcellulose (2-HEC, Sigma Aldrich) molecular weight powder average 90000, 10g / liter of peptone for mycology (Sigma-Aldrich), 10 mg / liter of spores and mycelium of Beauveria bassiana strain CD 1123.

In the preferred embodiment, the procedure for preparing to obtain about 120 g of matrix at 16% by weight of 2-HEC provides (starting from conditions of sterility of the materials and instruments):

a) prepare 100 ml of sterile distilled water,

b) add 1 g of peptone and mix at room temperature until completely dissolved,

c) add 1 g of conidia and mycelium of Beauveria bassiana strain CD 1123 (corresponding to a turbidity of 2McFarland and an initial concentration of about 10 <3> conidia / ml of suspension),

d) mix at room temperature for 10 minutes,

e) add 16 g of 2-HEC and mix at room temperature for 40 minutes (estimated time for the complete gelling of the 2-HEC which takes place through the hydration reaction. The time was verified by analyzing the viscosity variation over time under conditions standard).

The same preparation was performed in the experiments in the BB-free or peptone-free forms. In the description of the experiments, these alternative compositions will be referred to respectively as "prepared according to example 1 but without BB" and / or "prepared according to example 1 but without peptone".

These preparations all have pH values between 6-7 and salinity values below 3% under standard conditions (measured with a Mettler Toledo model pH8 benchtop pH meter and a Bormac COND 70+ portable conductivity meter, respectively).

Tests carried out

Tests have been carried out that aim to demonstrate that the matrix prepared as reported in example 1 works against the tiger mosquito (ie it simultaneously satisfies the functions of "biomimetic lure", "survival and growth of the biocide" and "kill") and that the values of the parameters necessary for the operation of each function are satisfied and are within the range of the more general ones reported in the description of the invention.

To evaluate the "lure" functionality, an oviposition test was carried out on the matrices made as in example 1 as the 2-HEC concentration varies. To evaluate the "survival and growth of the biocide" functionality, biocompatibility and conidic growth tests were carried out on matrices prepared as in example 1 (using the concentrations that were found to be most captivating in the deposition test). The growth test was combined with the lethality test on tiger mosquito eggs (Aedes albopictus) to evaluate the "kill" functionality. The test was performed at the end of the growth period of the Beauveria bassiana within the matrix prepared according to example 1. Tests were performed to evaluate viscosity, percentage of free water, yield stress. The values obtained guarantee on the one hand that the preferred preparation shown in example 1 falls within the parameters that define the invention and also that the values relating to the preferred preparation are really effective against the tiger mosquito.

Materials and methods and results

Measurement of the viscosity of the matrix via

The matrix prepared as reported in example 1 with weight concentrations from 2 to 30% of 2-HEC but without Beauveria bassiana was subjected to a test to evaluate its viscosity. The test is of the "single shear rate" type performed with a Malvern Kinexus pro rotational rheometer in flat-flat configuration. The test was performed with a constant shear rate equal to 50s <-1> for 5 minutes at 30 ° C and atmospheric pressure on each concentration of 2-HEC tested. The viscosity values varying the concentration by weight of 2-HEC (2-30%) at 30 ° C and atmospheric pressure are shown in figure 2. The results show that the functionalities that require a viscosity value of not less than 2 Pas are guaranteed by weight concentrations of 2-HEC not lower than 4%.

Measurement of the amount of free water present in the matrix

By free water we mean water that is not chemically linked to the material and therefore can freeze and melt at the characteristic temperatures of water and is the first to evaporate in environmental conditions. The test was performed with differential scanning calorimetry (DSC) with the Q2000 Series instrument by TA Instruments, on matrices with 2-HEC concentrations in the range 2-30% by weight prepared as in example 1 but without Beauveria bassiana . The test (performed in air) includes a cooling ramp at 5 ° C / minute down to - 20 ° C, an isotherm at -20 ° C for 10 minutes and a heating ramp at 5 ° C / minute up to 50 ° C (atmospheric pressure). From the analysis of the endothermic melting peaks it is possible to obtain the amount of free water inside the material. In particular, knowing the energy necessary for the fusion of a known quantity of water and peptone, it is possible to obtain the quantity of free water by analyzing the energy associated with the endothermic peak (area under the curve measured with the specific functionality of the software associated with the instrument). The same procedure was carried out on a masonite sample immersed in water for 48 h. Masonite was used as the lower limit of free water necessary for the deposition contained on a standard substrate which is certainly attractive. The results are shown in Figure 3 and show, as the concentration by weight of 2-HEC in the matrix prepared according to example 1 varies, the values of the free water contained in percentage with respect to the weight of the sample. The same figure also shows the value of free water contained in a sample of wet masonite. It is noted that as the concentration increases, the free water content decreases. For the matrix with a concentration of 16%, free water is 78% of the total weight of the sample.

Yield test

The yield test is used to measure the maximum yield stress value, that is the tangential stress value at which a fluid begins to flow. It was performed with a Malvern Kinexus Pro rotational rheometer in the plate-plate configuration at a temperature of 30 ° C and atmospheric pressure. The threshold value was calculated as that tangential stress value to which a 5mm layer of matrix is subjected if placed on a vertical wall, when subjected exclusively to its own weight. If the yield stress peak value is lower than the threshold value then the matrix placed on the vertical wall will leak.

The tests were performed as the concentration of 2-HEC by weight increases on matrices prepared according to example 1 but without Beauveria bassiana to avoid bad readings by the instrument. The absence or presence of BB does not involve significant changes in the yield test values.

The curve in figure 4 shows the yield stress values as the concentration by weight of 2-HEC of the matrix prepared as in example 1 increases. It is noted that the threshold value is exceeded starting from concentrations higher than 8% by weight of 2 -HEC calculated so that a 5mm thick layer does not run if spread on the vertical wall. Therefore, the 16% matrix made as in example 1 complies with the requirements of the various functions.

Weight loss test over time

The weight loss test was performed in a Binder model KBF 115 climatic chamber at 25 ° C and 70% relative humidity. In the test, the weight of a sample subjected to controlled environmental conditions is monitored daily. In the case of the matrix, the weight change is associated with the evaporation of free water. The matrix is attractive if after 15 days it has at least a quantity of free water equal to that of wet masonite (substrate classically used for deposition) which is between 30 and 50% by weight (in this specific case it is equal to 35% ).

The matrix prepared as in example 1 was spread on the walls of a trap like the one shown in figure 1. The trap and matrix system was weighed daily and the net weight of the matrix was obtained. Figure 5 shows the values of the normalized weights of the matrix (on the initial weight) with respect to time. We can see the weight variation or the loss of free water over time (the weight lost is associated with the evaporation of free water). The test shows how, even after 24 days, there is a weight of approximately 30% of the initial weight of the matrix which, starting from a matrix prepared as in example 1 which has 80% of free water, means having a quantity of free water equal to about 34% of the total weight of the matrix and therefore sufficient to be attractive since it is comparable to the limit condition established by the masonite.

After 15 days the matrix retains about 54% of the initial weight corresponding to a percentage of free water equal to about 60%, which is higher than the limit quantity defined by the humid masonite.

In addition, figure 5 shows the weight variation curve over time of the wet masonite (kept submerged until equilibrium is reached or the impossibility of absorbing further water). It is noted how the weight loss due to water evaporation is much faster in the masonite than in the gel.

Oviposition test for evaluation of biomimetic "lure" functionality.

One of the purposes of the matrix is to mimic a natural habitat of the tiger mosquito. The attractiveness of the matrix prepared as in example 1 is obtained from the test as the concentration of 2-HEC varies and the correlation between the characteristics of the substrate and the attractiveness is verified.

For the purposes of the test, a colony of tiger mosquitoes was reared in an insectarium placed at 26 ° C and 70% relative humidity and with a ratio of hours of light: hours of darkness 14:10. The mosquitoes had a blood meal 48 hours before laying (time needed for the eggs to mature). At the end of the maturation time, 30 pregnant females were kept in the cage. Inside the cage, 2 plastic containers were placed with 30 ml of distilled water. The control (blotting paper, C) was placed on the inner walls of the first container; the matrix prepared as in example 1, but without Beauveria bassiana (T), was manually spread on the walls of the second container. After 24h the containers were recovered and the eggs counted. The test was repeated 3 times for each 2-HEC concentration tested.

The values of the number of eggs laid on the matrix as the concentration of 2-HEC vary in the histogram of Figure 6. The reported value T / C represents the ratio between the number of eggs laid on the tested matrix T and the number of eggs laid on the standard control C (damp absorbent paper). When the T / C value is greater than 1 then the matrix is more attractive than the control. The curve of figure 6 instead shows the value of free water contained in the matrix used and the link between the free water parameter and the number of eggs laid on the matrix is highlighted with the definition of an optimal range of 2-HEC concentration. The results show that the best deposition results are obtained between 70% and 85% of free water or for concentrations between 8% and 20% by weight of 2-HEC and in particular the composition with 16% of 2-HEC (78% free water) is the most attractive.

However, the matrix has yield stress values that make it possible to use it on a vertical wall only starting from 10% of 2-HEC as the yield stress value is higher than the threshold value shown in Figure 4.

Biocompatibility and lethality test (evaluation of "survival and growth" and "kill" functions)

Since the matrix prepared as reported in example 1 must perform the "survival and growth of the biocide" function but also the "kill" function, two tests were performed in series: a biocompatibility and growth test of BB in the matrix and an efficacy test on tiger mosquito eggs (carried out using the matrices following the growth test of BB). The tests were performed on the matrix prepared as in example 1 at the concentration of 2-HEC which recorded the best result in terms of attractiveness in the oviposition test.

Biocompatibility and growth test of Beauveria bassiana in matrices

The test evaluates, through conidic counting, the impact on the vitality and growth of the biocide by the biopolymer used, the process of making the matrix and the microenvironment created by the matrix itself. The conidic count is used as the conidia are the infecting part of the entomopathogenic fungus therefore: the higher the conidic concentration the higher the infectious power.

The test was performed on the matrix with the concentration of 2-HEC which obtained the best result in terms of oviposition (16% of 2-HEC by weight) prepared as in example 1 and containing Beauveria bassiana. A 16% 2-HEC preparation containing 1% by weight of peptone and one without peptone were tested. The two types were compared with two suspensions based on distilled water (called G and H respectively) containing conidia and mycelium of Beauveria bassiana at the same initial concentration present in the matrix (shown in example 1). G contains 1% by weight of peptone while H lacks it.

In this way, the test also evaluates the effect, with the same concentration of 2-HEC, of the presence of the peptone as a nutrient for Beauveria bassiana.

All matrices and control solutions are stored in sterile glass containers in a Binder model KBF 115 climate chamber at 28 ° C and 80% relative humidity for 24 days (period comparable to the expected service time for the matrix ).

During this period a conidic count was carried out every 4 days by seeding on a plate for mycological growth based on potato dextrose agar (PDA) both on the matrices and on the suspensions. Conidic counting is performed with dilutions of the matrix in distilled water in the range 10 <-4> -10 <-1>. The "number of conidia-days" curves shown in Figure 7 compare the number of conidia of the entomopathogen Beauveria bassiana in the different growth media. The results show that, with the same concentration of 2-HEC, the conidic charge is constantly lower in matrices without peptone. Furthermore, it is noted that for suspensions, with and without peptone, the conidic charge is already lower than that of the matrices starting from day 8. Therefore the matrix prepared as in example 1 not only satisfies the "survival and growth" functionality but amplifies the growth of BB compared to a standard aqueous suspension.

Efficacy of the matrix on tiger mosquito eggs (Aedes albopictus) for different egg-matrix contact times

The same matrices used in the growth experiment and stored for 24 days under controlled conditions and comparable with those of use in the field of the matrix (temperatures 18-30 ° C and relative humidity higher than 70%), were used to test the effectiveness of the system against tiger mosquito eggs after 5, 10 and 15 days of egg-matrix contact. The term of comparison was the same suspensions of BB prepared for the growth experiment (G and H) while distilled water was used as a control which, having zero mortality, was not shown in the graph in figure 8. As further control, a solution of water and peptone 1% by weight was used to evaluate any effects on hatching (K).

The concentration of Beauveria bassiana inside the matrices that will act against the eggs will be that reached after 24 days of incubation, i.e. for matrices of the order of 10 <7> conidia / mg, while 10 <5> conidia / ml for the suspensions. Therefore, quantities of matrix and suspension will be used such as to have the same number of starting conidia.

The purpose of the test is to verify the "kill" functionality after verifying the "survival and growth" function.

All tests were carried out under sterile conditions on 5-day-old tiger mosquito eggs collected at the insectarium of the La Sapienza University of Rome.

To carry out the test, 1g of each matrix was spread on sterile filter paper (4x4 cm <2> and 1mm thick) while 1.5ml of each suspension (G and H and K in figure 8) and control based of distilled water were collected and uniformly dispersed on the surface of the sterile filter paper.

Three sterile papers were prepared for each matrix and suspension, one for each contact time (5, 10 and 15 days). On each of which 30 tiger mosquito eggs are placed. From this moment (T0) 5, 10 and 15 days of egg-matrix contact are counted.

During contact, the matrices are stored in a Binder model KBF 115 climatic chamber (separated from any contaminants and carefully cleaned) at a temperature of 28 ° C and relative humidity of 80%.

To evaluate the effectiveness, the samples thus prepared were subjected to a hatching test.

The same type of analysis was carried out on matrices prepared as in example 1 with concentrations of 2-HEC by weight ranging from 2-30% but without Beauveria bassiana as an additional evaluation of the effect of the matrix without Beauveria bassiana.

Hatching test

After the expected contact time (5, 10, 15 days), the papers with the matrix and the eggs are moved with disposable sterile forceps in sterile plastic containers filled with 80 ml of distilled water and 1 mg of yeast (necessary to favor hatching by reducing oxygen in the water and as a source of nourishment for the larvae). The maps with the matrix and the eggs are kept in water for 5 days (average hatching time for the eggs) in an air-conditioned environment at 25 ° C (the temperature and humidity are monitored with the aid of a data logger).

At the end of the 5 days, the presence of viable larvae within the hatching water is assessed by means of a larval count. In addition, with the aid of a Leica series 230 and model 238 optical microscope, any larvae trapped inside the matrix are searched for and the presence of Beauveria bassiana structures on the eggs is evaluated with a SEM EVO 40 electron microscope from Zeiss. Non-hatched eggs (which are monitored for a further 10 days) and non-viable larvae trapped in the matrix are considered non-viable.

The vitality of the eggs was preliminarily evaluated on the batch of eggs used for the test by carrying out a random hatching test on a group of 100 eggs performed with the same protocol reported.

The same type of test was performed on matrices prepared as in example 1 but without Beauveria bassiana with a concentration range of 2-30% by weight of 2-HEC and each containing 1% by weight of peptone in order to evaluate the effect itself of 2-HEC at various concentrations.

The histogram in figure 8 shows the corrected mortality values, evaluated as the number of viable larvae on the number of eggs placed on the matrix after 5, 10 and 15 days of egg-matrix contact. The reference control against which the corrected mortality was assessed is distilled water (0% mortality, not shown in the figure).

The yield stress curves and the threshold yield stress (i.e. the value above which the matrix can be used because it does not run) are also reported.

The results obtained shown in figure 8 are the average of 3 experiments, showing how for the matrices in which the peptone is present the mortality increases with the contact time while in the others it decreases. In the preferred form of preparation the corrected mortality is higher than 70% already at 10 days of contact. Therefore the matrix satisfies the functions of "survival and growth of the biocide" and "kill".

In the samples without BB tested the results shown in figure 9 show that all cases satisfy the “kill” functionality. For the matrices with concentration between 2-8% of 2-HEC the corrected mortality is between 80-90% but, by analyzing the matrices before the hatching test it was verified that the eggs were already hatched inside the matrix and are death before the hatching test, therefore this event cannot be associated with a direct action of the matrix. Furthermore, it can be seen that between 2-8% of 2-HEC the yield stress value does not exceed the threshold value and therefore the matrix cannot be used on a vertical wall as required by the "lure" functionality

For matrices at 16 and 30% of 2-HEC there is a corrected mortality that reaches 90% for the 15 days of egg-matrix contact. At these concentrations the eggs do not hatch before the test and therefore mortality is associated with the mechanical action of the matrix.

As an example, figure 10 shows the images taken (A) with a Leica series 230 model 238 stereo microscope showing the entrapment action of the larvae by the matrix on newborn larvae (mechanical action) and (B) obtained with a scanning electron microscope (SEM EVO 40 by Zeiss) which shows the action of BB on the eggs that are colonized by the mycelium of the fungus.

In figure 10A the arrow indicates an example of a larva trapped immediately after hatching and dead as it is unable to move to find food and / or breathe. In image 10B you can see the presence of the BB structures on the mushroom.

Example 2: preparation of a matrix for the control of the tiger mosquito (Aedes albopictus) based on 2-HEC and Beauveria bassiana subjected to a cold freeze-drying process.

An alternative form of preparation provides a matrix intended for the control of the tiger mosquito (Aedes albopictus) with the following composition: distilled water, 20-300g / liter of 2-hydroxyethylcellulose powder (2-HEC, Sigma Aldrich) average molecular weight 90000, 10g / liter of peptone for mycology (Sigma-Aldrich), 10mg / liter of spores and mycelium of Beauveria bassiana strain CD 1123.

The protocol for the preparation of 120g of matrix at 16% by weight provides for 2-HEC (starting from the sterile conditions of the materials and instruments):

a) prepare 100ml of sterile distilled water,

b) add 1g of peptone and mix at room temperature until completely dissolved,

c) add 1g of conidia and mycelium of Beauveria bassiana strain CD 1123 (corresponding to a turbidity of 2 Mc Farland and an initial concentration of about 10 <3> conidia / ml of suspension),

d) mix at room temperature for 10 minutes,

e) add 16g of 2-HEC and mix at room temperature for 30 minutes (estimated time for the complete gelling of the 2-HEC which takes place via hydration reaction. Time is verified by analysis of the viscosity variation over time), f) pour or inject the matrix thus prepared into a mold of the desired shape (for example in the cavity of a hollow cylinder with an internal radius of 10 cm and an external radius of 11 cm),

g) freeze the matrix and the mold for 2 hours at -40 ° C,

h) subject the frozen matrix to a freeze-drying process at 0.3 milibars and -40 ° C temperature for 12 hours until the complete elimination of the water by sublimation.

Biocompatibility test and conidic count on matrix subjected to freezedrying

A certain quantity of matrix prepared as in Example 2 was removed and rehydrated until a matrix at 16% by weight of 2-HEC was obtained, that is, until a matrix with the same characteristics as that of Example 1 was obtained. was incubated under the same conditions as the matrices made and tested in example 1, in order to assess whether the freeze-drying process had an impact on any of the matrix functions and in particular on that of "survival and growth". It emerged that the process does not affect the growth of Beauveria bassiana in the matrix, in fact after 24 days the concentration of the biocide is at the same level as an untreated matrix, prepared as in example 1 (Figure 11).

Another part of the matrix prepared as in example 2 was taken from the same preparation and stored in a closed container at room temperature. Every 4 days a quantity of matrix useful to perform the conidic count test was taken as previously described in example 1. From the test it emerged that the lyophilized matrix maintains a constant conidic concentration for the entire duration of the test, therefore it can be preserved without losing vitality and therefore effectiveness (figure 11).

Therefore the freeze-drying process does not alter the functioning of the matrix.

Example 3: preparation of a matrix for the control of the tiger mosquito (Aedes albopictus) based on 2-HEC cross-linked with polyethylene glycol-diglycidyl ether (PEGDE) and sodium hydroxide (NaOH).

One form of comparative preparation provides a matrix intended for the control of the tiger mosquito (Aedes albopictus) with the following composition: distilled water, 20-300 g / liter of 2-hydroxyethylcellulose (2-HEC, Sigma Aldrich) powder average molecular weight 90000, 10g / liter of peptone for mycology (Sigma-Aldrich), 20g / liter of NaOH, 20g / l of PEDGE.

The following procedure is aimed at preparing approximately 108g of cross-linked material, or a gel with 8% by weight of 2-HEC (if you want to reduce or increase the final quantity, reduce or increase all quantities proportionally).

1. Preparation of 100 g of a 0.5M NaOH solution (Solution A)

to. Weigh 2g of solid NaOH (0.05 moles).

b. Weigh 100g of distilled water into a suitably sized beaker.

c. Add the NaOH to the solution until completely dissolved (clear solution).

2. Preparation of 100g of a 0.5M HCl solution (Solution B, to be used at the end of the process to neutralize and wash the cross-linked gel).

to. Weigh 4.94g (or take 4.10ml) of 37% hydrochloric acid.

b. Add the HCl to 96g of distilled water and mix until the acid is completely dissolved.

3. Preparation of the crosslinking solution (Solution C)

to. Weigh 2g of PEGDE into a suitably sized beaker

b. Add the 100g of solution A and mix until completely dissolved (clear solution).

4. Preparation of the gel

to. Place the beaker containing solution C under a mechanical stirrer equipped with a suitable shovel. b. Stir the mechanical stirrer at a suitable speed

c. Weigh 8g of 2-HEC and 1g of peptone and add them to the solution under constant stirring

d. Incubate at a controlled temperature of 30 ° C for 5 hours 5. Wash the gel

to. Remove the cross-linked gel from the beaker, cut it into pieces. b. In a suitably sized beaker, weigh 400g of distilled water

c. Add 100g of solution B to obtain final 500g

d. Dip the gel into the washing solution and keep it stirring constantly for 2-3 days.

Oviposition test

A colony of tiger mosquitoes was raised in an insectarium placed at 26 ° C and 70% relative humidity and with a ratio of hours of light: hours of darkness 14:10. Mosquitoes had a blood meal 48h before laying (time necessary for the eggs to mature. At the end of the maturation time, 30 pregnant females were kept in the cage. 2 plastic containers with 30 ml of distilled water. The control (absorbent paper, C) was placed on the inner walls of the first container. The matrix (T) was positioned on the walls of the second container. After 24h the containers were recovered and the eggs were cashed. The test is was repeated 3 times with matrices having different concentrations of 2-HEC prepared as in example 3.

The histogram shows the values of T and C (already described above). The test shows how the presence of substances useful for crosslinking acts as a deterrent for egg laying. Specifically, residues of hydrochloric acid and / or NaOH and / or PEGDE are repellent for the deposition of the tiger mosquito.

In fact, from the results shown in figure 12 it emerges that the cross-linked gel described in example 3 is not attractive, on the contrary, it is totally rejected by insects. Failing the "lure" functionality the matrix is not functional.

Example 5: preparation of a matrix based on 2-hydroxyethylcellulose (2-HEC) and sorbitol, intended for the control of the tiger mosquito (Aedes albopictus).

According to this embodiment, the matrix is intended for the control of the tiger mosquito (Aedes albopictus) and has the following composition: distilled water, 20-300 g / liter of 2-hydroxyethylcellulose powder (2-HEC, Sigma Aldrich) average molecular weight 90000, 60g / liter of sorbitol (D-Sorbitol, Sigma-Aldrich) molecular weight 182.17.

In the preferred embodiment, the procedure for preparing to obtain about 120 g of matrix at 16% by weight of 2-HEC provides (starting from conditions of sterility of the materials and instruments):

a) prepare 100 ml of sterile distilled water,

b) add 6 g of D-Sorbitol and mix at room temperature until completely dissolved,

c) add 16 g of 2-HEC and mix at room temperature for 40 minutes (estimated time for the complete gelling of the 2-HEC which takes place via hydration reaction. The time was verified by analyzing the viscosity variation over time under conditions standard).

This preparation has pH values between 6-7 and salinity values below 3% under standard conditions (measured respectively with a Mettler Toledo model pH8 benchtop pH meter and a Bormac model COND 70+ portable conductivity meter).

In this preparation, the presence of the humectant D-Sorbitol in a concentration by weight of 6% reduces the evaporation rate while maintaining the conditions suitable for carrying out the various functions (related to the amount of free or bound water) for a longer time.

A tiger mosquito deposition test has been set up in the laboratory. In the test, the matrix prepared as in example 5 was compared with another, prepared as in example 5 but without D-sorbitol. Both were exposed to conditions suitable for spawning (25 ° C and 70-80% relative humidity). In the test, the number of eggs present on the matrix at that time was monitored weekly.

The test showed that both matrices are capturing. On the matrix without sorbitol the number of eggs remained approximately constant after 3 weeks while on the matrix containing 6% of sorbitol the deposition continued until week 5, demonstrating the prolonged maintenance of the conditions suitable for the lure.

Claims (11)

CLAIMS 1. Biomimetic insecticide matrix in the form of gel for bloodsucking insects having at the same time the functionality of "biomimetic lure and kill" and that of survival and growth of biocides inside it active against bloodsucking insects; said matrix comprising one or more biocompatible and biodegradable polymers selected from: agar, pectins, polysaccharides, chitosans, alginates, celluloses such as hydroxymethyl- and hydroxyethylcelluloses, carboxymethyl- and carboxyethylcelluloses and possibly humectants said matrix having a pH comprised between 4 and 8, salinity not higher than 3%, a quantity of free water higher than 0, preferably comprised between 5-98% by weight and a viscosity comprised between 10 <-3> -10 <4> Pas. 2. Insecticide matrix according to claim 1, further characterized by having yield stress values between 0-2000Pa, storage modulus and / or loss modulus values between 0-2MPa. Insecticidal matrix according to any one of claims 1-2, which maintains at least 30% of the initial weight within the fifteenth day of exposure to conditions of temperatures of 18-30 ° C and humidity of not less than 70%. 4. Insecticidal matrix according to any one of claims 1-3, which is active against insects selected from: horseflies, horse flies, mosquitoes, including C. tarsalis, C. pipiens, A. aegypti, A. sierrensis, A. nigromaculis, A. albimanus, A. albopictus, bedbugs, papadaci, fleas, ticks, lice, mites including Dermanyssus gallinae, nematodes, annelids, their larvae and eggs. 5. Insecticidal matrix according to any one of claims 1-4, which further comprises a bioinsecticide selected from: Gram-negative and Gram-positive bacteria, actinomycetes, fungi, protozoan yeasts, algae and their spores, in particular Bacillus subtilis subsp. Krictiensis, Pseudomonas pyrocinia, Pseudomonas fluorescence, Gliocladium wirens, Trichoderma reesei, Trichoderma harzianum, Trichoderma hamatum, Trichoderma viride, Streptomyces cacaoi subspecies asoensis; or microorganisms such as Bacillus thuringiensis, or entomopathogenic fungi that belong to four main groups: Oomycota, including Lagenidium and Leptolegnia, Ascomycota, including Aspergillus, Beauveria bassiana, Metarhizium, Paecilomyces, and Zygomycota, including Conidiobolus, Entomniaga, Entomnia Furia, Massospora, Neozygites, Pandora, Zoophthora. 6. Insecticidal matrix according to any one of claims 1-5 in which the wetting agent is selected from: glycerin, propylene glycol, glyceryl triacetate sorbitol, xylitol, maltitol, polydextrose, natural extracts such as quillaia, compounds such as lactic acid or urea. Insecticidal matrix according to any one of claims 5-6 which has a Beauveria bassiana content of at least 10 <6> -10 <7> conidia / mg of matrix. An insecticidal matrix according to any one of claims 1-7, which is in the form of gel, spreadable paste or other hydrated or dehydratable and rehydratable moldable form. An insecticidal device comprising the matrix according to any one of claims 1-8. 10. Insecticidal device according to claim 9, usable inside closed places such as homes, offices, hospitals, and in open environments such as parks, neighborhoods or cities. 11. Insecticidal device according to claim 9, usable in the veterinary, zootechnical, industrial disinfestation fields and in large-scale control campaigns.