WO2019229102A1

WO2019229102A1 - Beneficial metschnikowiaceae yeast cells, fragments thereof, or substances produced thereby for animals

Info

Publication number: WO2019229102A1
Application number: PCT/EP2019/063902
Authority: WO
Inventors: Maria Isabel POZO ROMERO; Alfredo BENAVENTE MARTINEZ; Gaby VAN KEMENADE; Annette VAN OYSTAEYEN; Felix WÄCKERS; Hans JACQUEMYN
Original assignee: Biobest Group N.V.; Katholieke Universiteit Leuven
Priority date: 2018-05-30
Filing date: 2019-05-29
Publication date: 2019-12-05
Also published as: NL2021015B1

Abstract

Methods for improving the fitness of animals and methods for rearing animals, comprising feeding said animals with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, particularly comprised within a food composition, are provided herein. Advantageously, said yeast, particularly food compositions comprising said yeast, when provided to an animal, improves the growth, the development, the reproductive capacity, the health, the behaviour, the colony development rate, the flight activity and/or the foraging activity of said animals.

Description

BENEFICIAL METSCHNIKOWIACEAE YEAST CELLS, FRAGMENTS THEREOF, OR SUBSTANCES PRODUCED THEREBY FOR ANIMALS

TECHNICAL FIELD

The present invention provides compositions for improving animal fitness, health and/or behavior, particularly arthropod fitness, health and/or behaviour. The invention further provides methods which make use of these compositions.

BACKGROUND OF THE INVENTION

Probiotics and prebiotics have been credited with providing all kinds of health and performance benefits, not only to humans, but also to animals.

Economic growth has increased the demand for agricultural, livestock products and aquaculture products over the past decades putting pressure on these sectors to produce more with limited resources. Pro- and prebiotics are being used successfully in livestock and aquaculture practices to increase disease to improve the digestibility of nutrients, increase tolerance to stress, increase disease resistance (e.g. preventing and controlling enteric pathogens), and improve performance (including reproduction and growth).

Nevertheless, there remains an interest in developing new compositions which can improve fitness in animals used in these sectors to further increase productivity.

Arthropods are important to humans in many ways. For example, arthropods can be used in biological pest control, as decomposers or in the production of several human- made products such as wax, silk or ingredients for medications. Furthermore, several arthropods, such as crustaceans (e.g. shrimp, prawns, crabs, lobsters) and insects are cultivated for use as human food. However, the greatest contribution of arthropods to the human food supply is by providing pollination services, thereby ensuring the successful production of arthropod pollinated fruit-bearing crops.

Arthropod pollinators, in particular insects, play an important role in plant reproduction and ecosystem functioning, by providing plants with the benefits of crosspollination. Insects are the prime pollinators of most agricultural crops and wild plants. In entomophilous plants (comprising 87% of angiosperms), pollination by insects has been shown to improve crop yield, individual fruit quality and quantity, shelf-life, taste, nutritional composition and market value compared to self-pollination. As a result, pollinator abundance and richness are essential features for both agricultural productivity and the conservation of wild plant communities. In turn, plants provide the visiting insects with nectar and pollen as the main floral rewards. However, in Western Europe and many other parts of the world, intensification of traditional farming practices over the last century has led to impoverished landscapes that represent poor habitats for many flower dependent insects due to a lack of suitable floral resources. In addition, chemical pesticides used in intensive agricultural production have been shown to have strong direct and indirect negative effects on pollinators. For example, fungicides may influence the gut flora of arthropods and thereby affect the host’s health and/or ability to digest food. Finally, certain insect pollinators (bees) are susceptible to various diseases and pests (mites). It is believed that these three (often interacting) mechanisms are the main factors underlying the decline of insect pollinator communities worldwide.

Efforts are being made to counteract this trend and to sustain pollinator diversity and fitness. The health, behavior and numbers of the pollinating insects can be improved by, for example, increasing the quantity and quality of their habitats, increasing the public awareness, prohibiting use of pollinator-harming pesticides, supporting bee keeping, etc. However, the need remains to improve pollinator fitness and health in order to ensure future pollination of both cultivated crops and wild vegetation.

Arthropods also play an important role in agriculture as predators and parasitoids in biological pest control. Also in this context, improvement of biodiversity is important, as species-rich populations are more likely to control pests than poorer ones.

SUMMARY OF THE INVENTION

The invention generally relates to the use of Metschnikowiaceae yeast cells, more particularly Metschnikowia gruessii yeast cells, an ascomycetes yeast, in the rearing of animals and/or as a pre- or probiotic, and/or to improve the fitness of animals. The inventors have found that, particularly when fed to arthropods, more particularly pollinating arthropods or arthropods able to feed on pollen or nectar, these yeast cells positively affect the fitness of these arthropods, such as reduced larval death, increased number of workers/colony etc. It is postulated that a positive effect on health can be obtained with Metschnikowiaceae yeast cells on all animals, as have been observed for Metschnikowia gruessii yeast cells. Indeed, it has been found by the inventors that Metschnikowia gruessii yeast cells (of different strains) are capable of reducing pathogen load in arthropods. Reducing pathogen loads relieves the arthropods from impairments associated with infection by said pathogens. Thus, the contacting of Metschnikowiaceae yeast cells to arthropods allows a general improvement of applications which make beneficial use of arthropods. Accordingly, the present invention relates to methods for improving the fitness of animals, comprising feeding said animals with Metschnikowiaceae yeast cells, such as Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby. In particular embodiments, said method for improving the fitness of animals is a method for improving the growth, the development, the reproductive capacity, the health and/or the behaviour of said animals. In particular embodiments, said animals are colony-forming arthropods and said method is a method for improving the colony development rate. In particular embodiments, said animals are pollinating or nectar/pollen-consuming arthropods and/or flying arthropods and said method is a method for improving flight and/or foraging activity.

Further provided are methods for rearing animals which comprises contacting said animals with Metschnikowiaceae yeast cells, such as Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby.

In particular embodiments of the methods of the present invention, said animals are arthropods. In certain embodiments, said animals are pollen- or nectar-consuming arthropods. In certain embodiments, said animals are pollinating arthropods. In certain preferred embodiments, said animals are Hymenoptera, preferably bees or bumblebees.

Further provided herein are methods for cultivating a fruit-bearing crop, comprising the steps of:

- feeding one or more pollinating arthropods with Metschnikowiaceae yeast cells, such as Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby; and

- ensuring pollination of the flowering crop by the one or more pollinating arthropods. Further provided herein is the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as a prebiotic and/or a probiotic.

In particular embodiments, Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby are used as a prebiotic and/or a probiotic for improving the immune system and/or immune function of animals, reducing the number of gut parasites, promoting the build-up of healthy gut bacteria and/or absorption of nutrients, support the digestive health and/or gut barrier function, inhibiting growth of harmful gut bacteria, and/or reducing stress.

Further provided herein are Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby for use as a medicament. Further provided herein are Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby for use in the prevention or treatment of gastrointestinal diseases, auto-immune diseases, infections, eczema, allergies, lactose intolerance, high blood pressure, and/or heart diseases.

Further provided herein are food compositions for animals comprising at least 100 of Metschnikowia gruessii yeast cells. In particular embodiments, the food composition further comprises one or more of a carbohydrate source, a nitrogen source, vitamins, lipids, fats, and/or minerals. In particular embodiments, said carbohydrate source comprises one or more sugars selected from the list comprising sucrose, maltose, glucose, fructose and dextrose.

The application further provides Metschnikowia gruessii strains which are particularly suitable for the methods and uses provided herein, more particularly a Metschnikowia gruessii yeast strain as deposited under the accession number 57395 at the BCCM/MUCL culture collection, or variants thereof.

In particular embodiments of the methods, uses and food compositions provided herein, said Metschnikowiaceae yeast cells, such as but not limited to Metschnikowia gruessii yeast cells are yeast cells from the Metschnikowia gruessii yeast strain as deposited under the accession number 57395 at the BCCM/MUCL culture collection, or variants thereof. In particular embodiments of the methods, uses and food compositions provided herein, said animals are Hymenoptera, preferably pollinating Hymenoptera, more preferably bees or bumblebees.

BRIEF DESCRIPTION OF THE FIGURES

The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.

Figure 1. Average number of days (expressed as the mean value ± standard error) needed to produce the first worker per nest upon administering living M. gruessii (alive suspensions of M. gruessii, Mg T1 ), medium modified by M. gruessii with the cells present (Mg T2) or only inactivated M. gruessii cells (Mg T3) to bumblebees, compared to control treatment (i.e. not comprising M. gruessii).

Figure 2. Mean number of bumblebee workers per nest (expressed as the model- adjusted mean ± standard error) obtained upon administering living M. gruessii (alive suspensions of M. gruessii cells; Mg T1 ), medium modified by M. gruessii with the cells present (Mg T2) or only inactivated M. gruessii cells (Mg T3) to bumblebees, compared to control treatment (i.e. not comprising M. gruessii cells), after 5 weeks (upper panel) and 9 weeks of development (lower panel).

Figure 3. Female (number of queens plus workers, upper panel) and male fitness (lower panel) of colonies fed with living M. gruessii (alive suspensions of M. gruessii cells; Mg T1 ), medium modified by M. gruessii with the cells present (Mg T2) or only inactivated M. gruessii cells(Mg T3) to bumblebees, compared to control treatment (i.e. not comprising M. gruessii cells). Bars denote least-adjusted means ± standard error. Figure 4. Relative consumption of treatment diet (comprised within a first sugar water feeder) compared to the consumption of sterile 50% sugar water (SW) (second sugar water feeder). Each open circle, filled triangle or open square denotes the consumption of a given nest in week 6 of the experiment. Filled triangle facing upwards denotes the treatment diet in which colonies are fed with living M. gruessii (alive suspensions of M. gruessii cells; Mg T1 ), the open square denotes the treatment diet in which colonies are fed with medium modified by M. gruessii with the cells present, filled triangle facing downwards denotes the treatment diet in which colonies are fed with only inactivated M. gruessii cells and the open circle denotes control treatment diet.

Figure 5. Mean individual worker weight (in mg) obtained by weighting 100 random workers per hive at the conclusion of the trial (16 weeks after start-up) for control and M. gruessii treated colonies. Mg T 1 denotes colonies fed with living M. gruessii cells, Mg T2 denotes colonies fed with medium modified by M. gruessii with the cells present, Mg T3 denotes colonies fed with only inactivated M. gruessii cells and control denotes a control treatment (i.e. not comprising M. gruessii cells). Bar heights denote mean ± standard error.

Figure 6. Average mortality of female (white bars) and male (gray bars) O. bicornis bees per cage over a 2 week-period. Bees of both sexes were reared in artificial separate cages with control treatment or treatment with living M. gruessii cells in suspension (Mg T1 ), next to standard feeding conditions.

Figure 7. Colony development of colonies fed with control (white) or M. gruessii cells treated pollen (gray). Colony development was checked at the end of the experiment (week 8) by showing average brood (i.e. the sum of egg cups and all larval stages) per colony ( left panel), and number of workers per colony at (right panel). Heights of bars depict least adjusted means ± standard error (SE). A, B, a, and b denote means that are different at p< 0.05, for a given variable. Figure 8. Average probing time ( in seconds), of B. terrestris naive bees visiting artificial flowers containing either control sugar water or sugar water with living M. gruessii cells in suspension (alive suspensions of M. gruessii Mg T1 ).

Figure 9. Average number of workers per colony after 10 weeks of development for each M. gruessii strain (see X axes on top of figure), dosage (number of cells/mI - see X axes at the bottom of the figure) and way of administration (see figure legend). Bar height indicate least-squares means plus standard error bars.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, the preferred methods and materials are now described.

While potentially serving as a guide for understanding, any reference signs in the claims shall not be construed as limiting the scope thereof.

In this specification and the appended claims, the singular forms“a”,“an”, and“the” include plural references unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of" also include the term “consisting of”.

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/-1 % or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. In the following passages, different aspects or embodiments of the invention are described in more detail. Each aspect or embodiment so described may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to“one embodiment”,“an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or“in an embodiment” in various places throughout this specification may but need not necessarily refer to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

The inventors found that certain yeasts, when supplemented to the diet, are able to have a positive effect on various aspects of the general fitness of an animal, in particular of an arthropod. In particular, the inventors have found that Metschnikowia gruessii yeast cells, when provided to an animal, such as via a food composition, enhances rearing, health, fitness and/or behaviour of the animal. Indeed, the inventors have found that providing Metschnikowia gruessii yeast cells to bumblebees, in particular when added to a food composition (e.g. sugar solution or pollen) , increases the rate of development of workers, the number of workers, the female fitness, the brood per nest and/or the size and weight of workers, and/or decreases the overall mortality. Furthermore, the inventors have isolated a particular strain of Metschnikowia gruessii which was found to be particularly potent in evoking the effects described above when ingested by an animal, in particular an arthropod.

A first aspect relates to a method for rearing animals, particularly arthropods, and/or a method for improving the fitness of animals, particularly arthropods, comprising providing said animals, particularly arthropods, with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby.

The“Metschnikowiaceae yeast cells”,“Metschnikowia gruessif’ or“M. gruessif’ yeast cells when referred to as such, or in the context of providing an animal with Metschnikowia gruessii yeast cells herein, refers to either living, active yeast cells or inactivated, dead yeast cells of the clade of Metschnikowiaceae, preferably of the genus Metschnikowia, more particularly Metschnikowia gruessii. Preferably, the yeast cells are living, active yeast cells. Metschnikowia gruessii yeast is an ascomycetous yeast that typically occurs in West-Central Europe, the Iberian Peninsula and Balearic Islands, where it can be found associated with floral surfaces, floral nectar and flower- visiting insects like bumble bees (Hautmann 1924; Gimenez-Jurado 1992; Herzberg et al. 2002; Brysch-Herzberg 2004; Herrera et al. 2010; Pozo et al. 201 1 ). Under stringent culture conditions, M. gruessii may produce asci and ascospores and reproduce sexually (Gimenez-Jurado et al. 1995; Miller & Phaff 1998). In particular embodiments, the Metschnikowia gruessii yeast as envisaged herein is the Metschnikowia gruessii yeast strain with accession number 57395 (BCCM/MUCL culture collection), as elsewhere described herein. The references made herein to and features described for Metschnikowia gruessii are believed to apply also to other yeasts of the genus Metschnikowia, and to yeasts of the family of the Metschnikowiaceae.

The term“fragments” when referring to yeast cell fragments as used herein, refers to any part of a yeast cell, including other organisms residing in the yeast cell, such as bacteria. Non-limiting examples of such fragments are the cell wall or a part of the cell wall or cell organelles, or constitutive parts of the cells such as nucleic acids or combinations thereof. Such fragments can be provided as extracts, homogenates or isolated components. In particular embodiments, the fragments represent the entire yeast and correspond to fragmented yeast cells.

The term “substances produced by a yeast” as used herein refers to substances produced by, and optionally secreted by a yeast cell, such as by Metschnikowia gruessii. Non-limiting examples of such substances include decomposition products, metabolites, enzymes, vitamins, sterols, and essential amino acids. For instance, the yeast metabolome database describes metabolites produced by the yeast Saccharomyces cerevisiae. Similarly, non-Saccharomyces yeasts have been shown to produce enzymes such as pectinase, protease, glucanase, lichenase, b-glucosidase, cellulase, xylanase, amylase, sulphite reductase, and lipase (Jolly et al, 2006, S. Afr. J. EnoL Vitie., VoL 27, No.1 : 15-39). Similar metabolites and/or enzymes can be identified for Metschnikowia gruessii. These metabolites may be produced during fermentation. Non-limiting examples of metabolites include lipids, sterols, vitamins, amino acids, peptides, organic acids, sugars, glycoproteins or derivatives thereof, organic esters, higher aldehydes and alcohols, vicinal diketones (VDK), sulfur volatiles. Substances produced by Metschnikowia gruessii yeast cells can be obtained by e.g. collection of the growth medium in which the Metschnikowia gruessii yeast cells have been cultured for at least 2 hours, preferably at least 6 hours, preferably at least 24 hours, 48 hours, or more, more preferably at least 24 hours. Accordingly, in particular embodiments, the substances produced by Metschnikowia gruessii yeast cells are comprised in cultivation medium. In particular embodiments, the growth medium in which the Metschnikowia gruessii yeast cells have been cultured is filtered with a filter having a pore size suitable for retaining Metschnikowia gruessii yeast cells and/or fragments thereof, resulting in medium comprising the substances produced by Metschnikowia gruessii yeast cells, but not Metschnikowia gruessii yeast cells or fragments thereof.

In particular embodiments, the substances produced by Metschnikowia gruessii yeast cells are one or more fatty acids.

In particular embodiments, the metabolites are collected as supernatant or extracts. In further embodiments, the metabolites are isolated metabolites.

In certain embodiments, a method for rearing animals, particularly arthropods, and/or a method for improving the fitness of animals, particularly arthropods, is provided, comprising providing said animals, particularly arthropods, with living, active cells or inactivated, dead cells of the yeast Metschnikowia gruessii, with fragmented cells of the yeast Metschnikowia gruessii, and/or with a (cell free) growth medium wherein the yeast Metschnikowia gruessii has been cultured or an extract thereof.

The term “rearing” as used herein, in the context of rearing animals, particularly arthropods, broadly refers to breeding and supporting growth, development, maintenance, and/or reproduction of animals. The term“rearing” may be used as a synonym for the farming of animals. The skilled person will understand that the rearing methods will differ for depending on the animal species. Suitable rearing or keeping methods for animals, particularly arthropods, are known to the skilled person. For example, bumblebees (e.g. B. terrestris) can be kept in a dark nest box under standard climatic conditions (e.g. 28°C and 60% relative humidity) and are typically fed ad libitum. The term“fitness” as used herein when referring to an animal or group of animals, particularly arthropods, is intended to refer to the ability of said animal or groups of animals (e.g. colonies or populations of animals) to survive and reproduce in the environment in which they find themselves. As a consequence of this survival and reproduction, the organism or group of animals will contribute genes to the next generation (Orr, Nature Reviews. Genetics, 10 (2009) 531-539.). Fitness comprises many different“fitness components” which contribute to the ability to produce viable progeny. As such,“fitness” also encompasses viability/longevity parameters, which are linked to general health and pathogen resistance, but also mating success, and fecundity (measured as daily fecundity or lifetime fecundity). For example, in colony- forming species, such as B. terrestris, realized fecundity of the mother queen will be apparent in the number of individuals (e.g., total number of workers and queen represents the“female fitness”, the total number of males represents the“male fitness”, the total number of sexuals (i.e. males and queens) represents the“colony fitness” and the total number of workers represents the “colony development”) present in the colony.

The term“fitness” may refer to physical and/or biological fitness, while both are usually linked to each other. Physical fitness is the physical ability to perform certain activities, such as flying, and may be evaluated by assessing the activity level, while biological fitness is the reproductive output, more particularly, the extent to which an animal is able to produce offspring in a particular environment. For colony forming arthropods, both physical and biological fitness can inter alia be evaluated by assessing the colony development and/or colony developmental parameters as described elsewhere herein. Accordingly, in particular embodiments, the method for improving the fitness of animals, particularly arthropods, and/or method for rearing animals, particularly arthropods as described herein involves or is a method for improving the biological fitness and/or the physical fitness of animals, particularly arthropods.

The term“providing” or“provide” as used in the context of the methods as described herein broadly refers to making the Metschnikowia gruessii yeast, as living active cells or dead, inactivated cells, fragments thereof or substances produced thereby, such as present in the growth medium wherein the Metschnikowia gruessii yeast has been cultured, available to the animal, particularly arthropods, for example by offering the Metschnikowia gruessii yeast, fragments thereof or substances produced thereby, as a food source, a food supplement, a prebiotic and/or a probiotic to said animal. In a preferred embodiment, providing in the context of the invention is intended to refer to orally administering the Metschnikowia gruessii yeast, fragments thereof or substances produced thereby. Oral administration to the animal allows for transfer of said Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby to the gut of the animal. Alternatively, the yeast, yeast fragments or yeast substances can be provided to the animal by positioning the yeast, yeast fragments or yeast substances, in the environment of the animal, so as to ensure direct physical contact of the animal therewith.

In particular embodiments, the Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby are provided to the animal, particularly arthropods, by oral administration.

The recitation “administered orally”, “oral administration”, “oral delivery”, “oral consumption”, or “administered per os” as used herein, refers to a route of administration to a subject where a substance or composition is taken through the mouth of said subject. One or more substances taken through the mouth may have a systemic or local (e.g. in the mount, oesophagus, stomach, intestines and/or rectum) effect in the subject. The dietary supplement may be ingested orally in the form of a powder, a tablet, a capsule, a pastille or a liquid preparation. If the dietary supplement is in powder form, it may be solubilized in any type of liquid, for example in water, milk or fruit juice, before administration to the subject.

In the context of the different aspects and embodiments of the present invention, the term“animal” as referred herein refers to eukaryotic and multicellular organisms. Most animals have a digestive tract, a nervous system, the ability to move voluntarily, and specialized sensory organs for recognizing and responding to their environment. Animals can typically not manufacture their own nutrition and must feed on plants, other animals, or other organic matter. Animals may be classified as vertebrates or invertebrates. Non-limiting examples of vertebrates are fishes, amphibians, reptiles, birds and mammals. Non-limiting examples of invertebrates are arthropods, molluscs, roundworms, ringed worms, and flatworms.

In particular embodiments, the animal is a mammal. The term“mammal” includes any animal classified as such, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, hamsters, rabbits, dogs, cats, guinea pigs, cattle, cows, sheep, horses, pigs and primates, e.g., monkeys and apes. In particular embodiments, the animal is a non-human animal. In particular embodiments, the animal is an arthropod, preferably a colony-forming arthropod. In certain embodiments, said animals are nectar- and/or pollen-consuming arthropods, i.e. arthropods who use nectar and/or pollen as a nutrient source. In more particular embodiments, said animals are pollinating and/or colony-forming insects, even more preferably said animals are bees or a bee of the genus Bombus (e.g. bumblebee).

In the context of the different aspects and embodiments of the present invention, the term“arthropod” as referred herein can be any arthropod from the phylum Arthropoda, including insects, arachnids, myriapods and crustaceans. Preferably, the arthropods are arthropods important as feed or food for animals, such as livestock, dairy animals, fish and/or humans, or are arthropods providing other products, such as silk, or services such as pollination and biological pest control. In certain embodiments, the arthropods as referred herein are nectar- and/or pollen-consuming arthropods, i.e. arthropods who are able to use nectar and/or pollen as a nutritional source. Such arthropods are thus able to be reared or sustained by supplying them with nectar and/or pollen. The skilled person understands that many arthropods are capable of using nectar and/or pollen as a food source, not only herbivorous arthropods, such as pollinating insects, but also predatory arthropods, such as predatory arthropods that are used as a biological control agent for the biological control of pests, e.g. predatory mites or parasitic wasps. In particular embodiments, the arthropods are insects, preferably nectar- and/or pollen-consuming insects, more preferably pollinating insects. Non-limiting examples of pollinating insects are bees, butterflies, moths, ants, wasps, flies, midges, mosquitoes or beetles. In particular embodiments, the arthropods are bees, preferably, bumblebees or honey bees, more preferably bumblebees of the genus Bombus. In particular embodiments, the arthropods are pollinating colony- forming insects. In particular embodiments, the insects belong to the order of Hymenoptera, such as to the suborder of Apocrita, more particularly to the superfamily of Apoidea. In particular embodiments, the arthropods belong to the family of Apidae. In further particular embodiments, the insects are from the genus Acamptopeum, Anthemurgus, Antherenoides, Acanthopus, Afromelecta, Agapanthinus, Aglae, Aglaomelissa, Alepidosceles, Alloscirtetica, Amegilla, Ancyla, Ancyloscelis, Anthophora, Anthophorula, Apis, Apotrigona, Arhysoceble, Austroplebeia, Axestotrigona, Bombus, Brachymelecta, Caenonomada, Camargoia, Canephorula, Cemolobus, Centris, Cephalotrigona, Chalepogenus, Chilamalopsis, Cleptotrigona, Coelioxoides, Ctenioschelus, Ctenoplectra, Ctenoplectrina, Cubitalia, Dactylurina, Deltoptila, Diadasia, Diadasina, Duckeola, Elaphrophoda, Epeoloides, Epicharis, Epiclopus, Eremapis, Ericrocis, Eucera, Eucerinoda, Eufriesea, Euglossa, Eulaema, Exaerete, Exomalopsis, Florilegus, Friesella, Frieseomelitta, Gaesischia, Gaesochira, Geniotrigona, Geotrigona, Habrophorula, Habropoda, Hamatothrix, Heterotrigona, Homotrigona, Hopliphora, Hypotrigona, Isepeolus, Lanthanomelissa, Leiopodus,

Lepidotrigona, Lestrimelitta, Leurotrigona, Liotrigona, Lisotrigona, Lophothygater, Lophotrigona, Martinapis, Melecta, Melectoides, Meliphilopsis, Meliplebeia, Melipona, Meliponula, Melissodes, Melissoptila, Melitoma, Melitomella, Meliwilea, Mesocheira, Mesonychium, Mesoplia, Micronychapsis, Mirnapis, Monoeca, Mourella, Nannotrigona, Nanorhathymus, Nogueirapis, Notolonia, Odontotrigona, Osirinus, Oxytrigona, Pachymelus, Pachysvastra, Papuatrigona, Paratetrapedia, Paratrigona, Paratrigonoides, Paepeolus, Pariotrigona, Partamona, Peponapsis, Platysvastra, Platytrigona, Plebeia, Plebeiella, Plebeina, Protosiris, Ptilothrix, Ptilotrigona, Rhathymus, Santioga, Scaptotrigona, Scaura, Schwarziana, Schwarzula,

Simanthedon, Sinomelecta, Sundatrigona, Svastra, Svastrides, Svastrina, Syntrichalonia, Tapinotapsis, Tapinotaspoides, Tarsalia, Teratognatha, Tetragona, Tetragonilla, Tetragonisca, Tetragonula, Tetralonia, Tetraloniella, Tetralonioidella, Tetrapedia, Tetrigona, Thygater, Thyreomelecta, Thyreus, Toromelissa, Trichocerapis, Trichotrigona, Trigona, Trigonisca, Trigonopedia, Ulugombakia, Xenoglossa, Xeromelecta, Zacosmia, Aethammobates, Ammobates, Biastes, Brachynomada, Caenoprosopina, Caenoprosopis, Chiasmognathus, Doeringiella, Epeolus, Hexepeolus, Holcopasites, Kelita, Melanempis, Neolarra, Neopasites, Nomada, Odyneropsis, Oreopasites, Parammobatodes, Paranomada, Pasites, Pseudepeolus, Rhinepeolus, Rhogepeolus, Rhopalolemma, Schmiedeknectia, Sphecodopsis, Spinopasites, Thalestria, Townsendiella, Triepeolus, Triopasites, Allodape, Allodapula, Braunsapis, Creatina, Compsomelissa, Effractapis, Eucondylops, Exoneura, Exoneurlla, Exoneuridia, Macrogalea, Manuelia, Nasutapis, or Xylocopa. Preferably, the arthropods are bees or bumblebees, in particular bumblebees of the genus Bombus, such as B. terrestris, B. ignitus, B. diversus, B. occidentalis, including related species and sub-species. In particular embodiments, the arthropod is B. terrestris; such as B. terrestris africanus, B. terrestris audax, B. terrestris calabricus, B. terrestris canadensis, B. terrestris dalmatinus, B. terrestris lusitanicus, B. terrestris sassaricus, B. terrestris terrestris and B. terrestris xanthopus. In particular embodiments, said arthropod is a biological control agent for the biological control of pests, as known by the skilled person, such as a predatory mites, a parasitic wasp or a predatory insect, such as a ladybug, hoverfly, lacewing or a Mirid bug. In particular embodiments the predatory mite is a member of the family of Phytoseiidae. Phytoseiid predatory mites are widely used for biological control of pests such as spider mites, thrips, and whitefly, particularly in greenhouse crops. In particular embodiments, the predatory mites are from a genus selected from the group consisting of Euseius, Amblyseius, Neoseiulus, Iphiseius, Indoseiulus, Kampimodromus, Typhlodromalus, Phytoseius and Typhlodromus

Without being bound by theory, it is believed that Metschnikowia gruessii yeast may provide certain components, such as decomposition products, and/or metabolites or substances, such as volatiles, monosaccharides or fatty acids, , to the animal to which it is provided, which improves the growth, the development, the reproductive capacity, the health, and/or the behaviour of the animal. Yeasts surviving in the gut of insects have been shown to provide decomposition products, B vitamins, sterols, or essential amino acids to their insect host (Jones 1984, Douglas 1998, Vega & Dowd 2005, Lee et al 2014). Therefore, it is reasonable to assume that the production of certain components, such as decomposition products, and/or metabolites or substances, such as volatiles, monosaccharides or fatty acids, by Metschnikowia gruessii yeast cells provides a fitness advantage for their hosts, and/or improves the growth, the development, the reproductive capacity, the health and/or the behaviour of the host, especially if the host is an animal which typically relies on nutritionally poor or unbalanced substrates (e.g. floral nectar or pollen). Metschnikowia gruessii yeast cells may help the host to digest its natural food source. Several lines of evidence indeed indicate that the nutrients from a food source, for example pollen, are not promptly available to the host and that some hosts (e.g. arthropods such as insects) are unable to consume particular types of unprocessed food sources, for example fresh unprocessed pollen. More particularly, for honeybees, it is known that they cannot digest starch well, such that they have difficulties digesting pollen types with a high- starch content.

Accordingly, the application provides methods for rearing animals, more particularly arthropods, which can be considered improved methods for rearing as the health of the animals is improved. In particular embodiments, the method for improving the fitness of animals, particularly arthropods as envisaged herein, or method for rearing animals, particularly arthropods as envisaged herein, is or involves a method for improving the growth, the development, the reproductive capacity, the health and/or the behaviour of animals. The effect of the yeast cells or products derived therefrom described herein can be ascertained on the individual animal or on a group of animals. More particularly where the effect is on reproduction and behavior and/or where the animal is small and reared in high numbers, the effect will be ascertained on a group of animals. In particular embodiments, the application envisages using the methods for improving the fitness of animals, particularly arthropods, in methods of rearing. Indeed, it is envisaged that there is an interest in contacting or feeding the animals, particularly arthropods, with the yeast cells and products of the invention during rearing so as to ensure a higher number/ higher survival rate of the offspring. In this context, reference can be made to a“group of animals”, particularly a“group of arthropods”.

The phrase“group of animals” as used herein refers to a number of animals of the same species. In particular embodiments, the group of animals are animals which by nature live and look for food together. Living in a group may have several benefits for the animals, such as group acquisition of food, access to mates, protection from predation, division of labour, and/or group defence. A group of animals may be a small group of animals (e.g. less than 1000 individuals), such as a group of horses, cows or sheep; or a large group of animals (e.g. more than 1000 individuals), such as a group of ants, bees or termites. Depending on the animal species living in a group, such a group of animals may be named differently. For example, a colony of bees is a synonym for a group of bees, a herd of bison is a synonym for a group of bison, a flock of birds is a synonym for a group of birds, a brood of chickens is a synonym for a group of chickens, and a shoal of fish is a synonym for a group of fish.

The term“growth” as used herein may refer to either the growth of a group of animals or arthropods, which is an increase in the total number of individuals present within the group of animals or arthropods, or to the growth of an individual animal, which is an increase in the size and/or weight of an individual animal or arthropod.

The term “development” as used herein when referring to an individual animal or arthropod, refers to the processes that lead eventually to the formation of a new individual starting from the fertilized egg. During development of an individual animal, the fertilized egg undergoes cell divisions to increase the number of cells; simultaneously, the cells produced differentiate into the organs and organs systems of the fully formed organism. The skilled person will understand that developmental processes may be different for different animal species. For example, a human may develop from a fertilized egg to an embryo, to a foetus, to a neonate, to an infant, to a child, to a young adult, to an adult, while a bee develops from a fertilized egg to a larva, to a pupa, to an adult bee.

The term“development” as used herein where referring to a group of animals or to colony development, refers to the advancement of a discrete group of animals, such as eusocial animals, of the same species living and/or growing together in a shared domicile. For example, a colony of colony-forming arthropods is a highly-organised animal society with cooperative brood care, overlapping generations within a colony of adults and a division of labour/tasks into reproductive and non-reproductive groups. In colonies of certain arthropods, there is one queen or single breeding female who produces the offspring and many workers that take care of the eggs and larvae, forage for food and/or protect the colony. For example, a bee or bumblebee colony is initiated by a mother queen laying fertilised eggs in a nest. The queen starts a colony by laying fertilised eggs that will develop into workers that will feed the following groups of offspring and take care of foraging. As the colony grows progressively larger, male bees/bumblebees and new queens are produced as well.

In particular embodiments, the methods for improving the fitness of animals, particularly arthropods, as described herein are methods for improving the developmental rate of animals, particularly arthropods. In more particular embodiments, the methods for improving the fitness of animals are methods for improving the colony developmental rate of colony-forming arthropods, preferably wherein said colony-forming arthropods are pollinating colony-forming arthropods. Similarly, also provided herein is the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby for improving the developmental rate of animals.

The phrase“developmental rate of (a group of) animals” or“colony developmental rate” as used herein refers to the speed at which the development of a group of animals or the colony development occurs. Typically, the term“rate” as used herein refers to a rate of change, preferably per unit of time. The rate of the development of a group of animals or the colony development rate can be assessed by different parameters known by the person skilled in the art. For example, the colony development rate of colony-forming arthropods could be evaluated by the time of first egg laying, the duration of development from egg to adult, or the number of individuals at a certain time interval, such as evaluating the size of the colony at time of sexual production, including evaluating all parameters relating to colony size, i.e. number of egg cups, larvae, pupae, workers, and sexuals (males and queens). The time interval or time of sexual production will differ depending on the developmental period of the type of colony-forming arthropod. An increase in the number of future workers, colony size and/or amount of brood within a certain time frame and/or when compared to a reference value is indicative of an improved colony development. Preferably, said reference value is the value obtained for a colony of arthropods not receiving the same treatment, i.e. not reared according to the methods according to present invention, or not having been provided with Metschnikowia gruessii, fragments thereof or substances produced thereby as described herein.

The term“reproductive capacity” as used herein refers to the ability to successfully generate progeny or offspring. An improved reproductive capacity of a group of animals, particularly arthropods, may lead to an increase in the size of the group of animals or arthropods, meaning that the total number of individual animals present within the group of animals increases.

As detailed above, in particular embodiments, the invention provides methods of rearing animals which comprise the step of contacting or feeding the animals, particularly arthropods with the Metschnikowia gruessii yeast cells, such as with living, active cells or inactivated, dead yeast cells, or products of the invention, such as yeast cell fragments or the (cell-free) growth medium wherein the Metschnikowia gruessii yeast cells have been cultured. In particular embodiments, the Metschnikowia gruessii yeast is the Metschnikowia gruessii yeast strain with accession number 57395 (BCCM/MUCL culture collection), as elsewhere described herein.

In particular embodiments, the animals are arthropods, such as nectar and/or pollen consuming or pollinating arthropods, and the method for improving the fitness of arthropods and/or for rearing arthropods is a method which involves and/or a method for improving the number of workers, the female fitness (fitness of the female workers and the queen), the size of workers, the weight of workers, and/or the brood (i.e. the number of egg cups and larvae) of a colony of arthropods.

Similarly, the invention provides for the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, as described above, for improving the number of workers, the female fitness (fitness of the female workers and the queen), the size of workers, the weight of workers, and/or the brood (i.e. number of egg cups and larvae) of a colony of arthropods.

The term“behaviour” as used herein, refers to the range of actions and/or responses made by organisms in conjunction with themselves or their environment (including other organisms and the physical environment). The behavior may be a reaction of the organism to one or more stimuli, such as internal or external stimuli. The reaction may be a conscious or subconscious reaction. The behavior of an animal is apparent from typical activities performed by the animal. The skilled person will understand that the type of activities will be different for different types of animals and between group- forming/colony-forming or solitary animals. Behavioral tests for determining the behavioral responses of an animal and methods for determining the behavior of an animal are known in the art. For example, for pollinating arthropods such as pollinating insects,“central place foragers” leave from their domicile/nest to go and search for food and return when they have succeeded in their search. Accordingly, the number of animals leaving or returning to the colony can be used as a measure for the general behavior or activity of the pollinating insect. Alternatively, flight behavior and foraging success can be used as a measure for the fitness of the pollinating (flying) insect, and, if the pollinating insect is a colony-forming insect, to study the colony fitness. A fit colony is characterized by a high number of pollinating insects making a high number of flights to gather food. An increase in flight activity and pollen/nectar collection will also lead to a more intense and a more efficient pollination of flowering crops. Non- limiting examples of other activities that can be studied to assess the behavior and fitness of colony-forming and/or pollinating insects are reaction to pheromones, temperature response, swarming behavior, running behavior, mating behavior and/or frequency, grooming and/or hygienic behavior, food storage behavior, guarding behavior and drifting behavior.

Accordingly, in particular embodiments, the method for improving the fitness of animals, particularly arthropods, as described herein, is a method for improving the flight and/or foraging activity of animals, preferably wherein said animal is a nectar or pollen consuming, pollinating and/or flying arthropod, more preferably wherein said animal is a bee or a bumblebee, even more preferably wherein said animal is a bee of the genus Bombus, in particular B. terrestris. Similarly, also provided herein is the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, as described herein, for improving the flight and/or foraging activity of animals. The term “flight activity” as used herein refers to the number of in- and out-flying pollinating arthropods and/or the frequency of in- and out-flying of pollinating arthropods from the nest over a certain period of time. The term“foraging activity” as used herein refers to the number of flowers visited per foraging flight and/or the amount of pollen or nectar collected. The term“health” as used herein, refers to the general well-being of an animal. The health of animal can be influenced by several environmental and/or genetic, or intrinsic and/or extrinsic, factors acting separately or in combination. For example, for arthropods these factors include the effects of intensive agriculture and pesticide use, starvation, malnutrition, viruses, attacks by pathogens and internal or external parasites and environmental changes. A decreased health will most often also affect the behaviour of the animal in a negative manner. The health of an animal can be assessed by, inter alia, size, weight, lifespan, reproductive output and resistance to infection with pathogens and parasites.

The application also provides the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, as described herein, as a probiotic and/or a prebiotic. In particular embodiments, the Metschnikowia gruessii yeast is the Metschnikowia gruessii yeast strain with accession number 57395 (BCCM/MUCL culture collection), as elsewhere described herein.

The term“probiotic” as used herein, refers to microorganisms, such as bacteria and yeasts, which exert beneficial effects on the health of the host, especially on the digestive system, when consumed in adequate amounts. Such health benefits may include, but are not limited to, preventing allergies; controlling high blood pressure; reducing cholesterol levels; treating gastroenteritis, inflammatory bowel disease and/or inflammatory bowel disease; preventing skin conditions such as eczema; and improving immune function.

The term“prebiotic” as used herein, refers to (typically non-digestible) food ingredients which beneficially affect the health of the host when consumed, more particularly through beneficial effects on microflora present within and/or ingested by said host. Such health benefits may include, but are not limited to, selective stimulation of the growth and/or activity of one or a limited number of beneficial gut bacteria, stimulation of the growth and/or activity of ingested probiotics, improved gut health, improved digestion, enhanced immune function, reduced risk for heart diseases, hormone regulation, improved bone health and increased resistance to invading pathogens.

In particular embodiments, the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as envisaged herein, is aimed at improving the immune system and/or immune function of animals, reducing the number of gut parasites, promoting the build-up of healthy gut bacteria and/or absorption of nutrients, support the digestive health and/or gut barrier function, inhibiting growth of harmful gut bacteria and/or reducing stress, preferably for supporting gut barrier function and/or promoting the absorption of nutrients.

The term“gut barrier function” as used herein refers to the ability of the digestive system to control absorption of nutrients within the intestine and to defend the body from dangerous macromolecule penetration. The role of the intestines or gut in protecting the mucosal tissues and circulatory system from exposure to pro- inflammatory molecules, such as microorganisms, toxins, and antigens is vital for the maintenance of health and well-being of the subject. The intestinal or gut barrier is a complex system composed of physical (e.g. mucous layer, intestinal epithelial cells), chemical (e.g. gastric acid, digestive enzymes), biological (e.g. normal intestinal flora), and immunological (e.g. lymphocytes) barriers. A disrupted intestinal mucosal barrier can allow passage of microbes, microbial products, and foreign antigens into the mucosa and the body of a subject. This can result in activation of the immune system and secretion of inflammatory mediators. Certain immune responses can subsequently cause cellular damage that can result in further barrier dysfunction. Defects in intestinal mucosal barrier function with the accompanying translocation of microbes and their products have been linked with a variety of health conditions. Non-limiting examples of health conditions in which intestinal mucosal barrier dysfunction is involved include food allergies, microbial infections, irritable bowel syndrome, inflammatory bowel disease, celiac disease, metabolic syndrome, non-alcoholic fatty liver disease, diabetes, and septic shock.

In particular, arthropods, and especially bees, seem to be particularly sensitive to gut parasites, such as Nosema bombi or Crithidia spp.. Ingestion of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby may improve the immune functioning of arthropods and reduce the number of gut parasites. Without being bound to theory it is believed that the impact of Metschnikowia gruessii yeast cells on parasites and pathogenic bacteria and other fungi can be due to competition processes, and also due to priority effects: the changes (pH, carbon and nitrogen sources that are metabolized, produced by the first arriving organisms (i.e. yeast) make the environment unsuitable for later arriving organisms.

Accordingly, also provided herein is the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby for improving immune functioning of arthropods, such as nectar- and/or pollen-consuming or pollinating arthropods, preferably wherein said Metschnikowia gruessii yeast cells decreases gut parasites such as Crithidia bombi. Further provided herein are agricultural methods, more particularly agricultural methods which involve animals, particularly arthropods, which comprise contacting or feeding said animals, particularly with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described herein. Indeed, all agricultural methods which involve animals can benefit from the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described herein to increase said animal fitness. In certain embodiments, the application provides a method for cultivating a crop which involves pollination and/or biological pest control by one or more animals, preferably one or more arthropods, more preferably one or more arthropods wherein the animals are contacted with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described herein above. In particular embodiments, the Metschnikowia gruessii yeast is the Metschnikowia gruessii yeast strain with accession number 57395 (BCCM/MUCL culture collection), as elsewhere described herein.

As the pollination of flowering fruit-bearing crops is for a large part dependent on pollinating insects, the skilled person understands that the presence of lower numbers of pollinating insects, or less active or less fit/healthy pollinating insects has a negative effect on the pollination of insect pollinated crops, resulting in a decreased number of pollinated flowers, and consequently a decreased number of fruits which can be harvested. Accordingly, by providing Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, optionally comprised within a food composition as envisaged herein, to an arthropod, particularly a pollinating insect, and thus increasing the health and/or fitness and/or behavior of the pollinating insect and/or (optionally) the colony development, the yield of fruit-bearing crops can be increased. Accordingly, in particular embodiments of the methods for cultivating a crop as described herein, the crop is a fruit-bearing crop, more particularly a flowering fruit- bearing crop.

The term“crop” as used herein refers to all cultivated plants or agricultural produce, grown for profit or subsistence. The term“fruit-bearing crop” as used herein refers to perennial edible crops, where the edible product is a true botanical fruit or is derived therefrom. Preferably, the term“fruit-bearing” refers to the potential of the crop to bear fruits, rather than to the actual status of the crop. The skilled person will understand that pollination of the fruit-bearing crop typically occurs at the blooming stage or flowering stage of the crop, which is before the fruit-bearing crop is actually carrying fruits. The term“flowering crop” or“blooming” as used herein refers to flower-bearing crops, more particularly, crops which require pollination to enable fertilization. Non- limiting examples are apple, pear, quince, sorbus, loquat, cherry, plum, apricot, almond, peach, strawberry, raspberry, oleaster, sea buckthorn, European walnut, pecan, hazelnut, pistachio, olive, persimmon, fig, mulberry, pomegranate, feijoa, tangerine, orange, lemon, grapefruit, citron, currant, gooseberry, European hazel, Actinidia, Schizandra, honeysuckle, viburnum, barberry, avocado, date palm, mango, breadfruit tree, papaya, banana, tomato, peppers, melon, cucumber, squash, beans, cotton, and the like.

The term“pollinate” or“pollination” as used herein refers to the process by which pollen is transferred to the female reproductive organs of a plant, thereby enabling fertilization to take place. For the process of pollination to be successful, a pollen grain produced by the anther, the male part of the flower, must be transferred to a stigma, the female part of the flower, of a plant of the same species. Pollination as referred herein is preferably cross-pollination, wherein the pollen from the anther of a flower on one plant is transferred to the stigma of the flower on another plant of the same species by a pollinating insect.

In particular embodiments, the method for cultivating a crop as described herein, comprises the steps of: contacting, (e.g. feeding) pollinating arthropods with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described herein and ensuring pollination of the flowers of said fruit-bearing crop by said pollinating arthropods. In particular embodiments, said method makes use of one or more colonies of pollinating arthropods.

Accordingly, in particular embodiments the methods will involve providing one or more pollinating arthropods which are reared according to the method for rearing a crop as described herein.

Where the biological pest control relies on the presence of arthropods, the skilled person understands that the presence of lower numbers of predatory arthropods, or less active or less fit/healthy predatory arthropods has a negative effect on biological pest control. Accordingly, in particular embodiments, the method for cultivating a crop as described herein involves biological pest control by one or more arthropods, preferably one or more arthropods reared according to the methods for rearing animals as described herein.

The term“pest” as used herein, refers to any organism which is detrimental to crops, for example by feeding on the crop. In particular embodiments, the biological pest are thrips, (spider) mites and/or whitefly. Exemplary thrips species include Western Flower Thrips (Frankliniella occidentalis) and Onion thrips (Thrips tabaci). Exemplary mites include two-spotted spider mites (Tetrancychus urticae), red mites (Dermanyssus gallinae), cured ham mites (Tyrophagus putrescentiae) and dust mites (Dermatophagoides farinae and Dermatophagoides pteronyssinus).

Also provided herein are methods for biological pest control comprising a step of providing predatory arthropods, such as mites, with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby. More particularly, the invention provides methods for biological pest control of a crop, said method comprising feeding one or more arthropods with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, wherein the one or more arthropods are a biological control agent for the biological control of pests, and contacting the one or more arthropod with the crop to ensure the biological control of pests on said crop. In certain embodiments, said method is a method for cultivating a crop of for biological pest control and comprises the steps of feeding one or more arthropods with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, wherein said one or more arthropods is a predatory arthropod or a biological control agent for the biological control of pests; and

- contacting the one or more arthropod with the crop to ensure the biological control of pests on said crop. In particular embodiments, the Metschnikowia gruessii yeast is the Metschnikowia gruessii yeast strain with accession number 57395 (BCCM/MUCL culture collection), as elsewhere described herein.

Similarly, also provided herein is the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby for improving the cultivation of crops, for example, by improving the pollination and/or biological pest control.

The skilled person will understand that the methods described herein which increase the fitness of animals are also suitable for increasing the production of products derived or obtained from said animals, such as meat, wool, leather, milk, egg and/or honey.

Accordingly, the application also provides methods for producing animal-derived products comprising a step of providing animals with a Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby so as to increase the fitness of said animals.

More particularly, the skilled person will understand that when the animals as referred to herein are honey bees, the methods for improving the fitness of honey bees as described herein, such as increasing the flight activity of honey bees as described herein, will lead to an increase in nectar collection and consequently an increase in production of honey by said honey bees.

Accordingly, in particular embodiments, the methods for the production of animal- derived products include methods for producing honey comprising a step of providing honey bees with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby so as to increase the fitness of said honeybees.

Similarly, also provided herein is the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby for improving the production of animal-derived products.

A further aspect relates to Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby or a composition comprising Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described elsewhere herein for use as a medicament. Indeed, it is envisaged that the present compositions which increase the fitness of animals can be used not only to improve the fitness of healthy animals but also have a therapeutic effect on diseased animals and will aid in the prevention of certain diseases. The treatment and prevention of different diseases is envisaged. More particularly the treatment and prevention of diseases which are linked to metabolic disorders are envisaged.

More particularly the application relates to Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby or a composition comprising Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described elsewhere herein for use in the prevention or treatment of gastrointestinal diseases, auto-immune diseases (e.g., rheumatoid arthritis), infections (e.g. vaginal infections, ear infections, throat infections), eczema, allergies, lactose intolerance, high blood pressure, and/or heart diseases, preferably gastrointestinal diseases and/or infections in a subject. Except when noted, the terms“subject” or“patient” can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred subjects are human subjects. More preferred subjects are adult human subjects.

The terms “treat” or“treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of gastrointestinal diseases, auto-immune diseases (e.g., rheumatoid arthritis), infections (e.g. vaginal infections, ear infections, throat infections), eczema, allergies, lactose intolerance, high blood pressure, and/or heart diseases, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of gastrointestinal diseases, auto-immune diseases (e.g., rheumatoid arthritis), infections (e.g. vaginal infections, ear infections, throat infections), eczema, allergies, lactose intolerance, high blood pressure, and/or heart diseases. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms, diminishment of extent of disease, and the like.

In particular embodiments, said gastrointestinal disease is selected from the list consisting of irritable bowel syndrome, inflammatory bowel disease, infectious diarrhea, and antibiotic-related diarrhea.

Also provided herein are methods of treating or preventing gastrointestinal diseases, auto-immune diseases (e.g., rheumatoid arthritis), infections (e.g. vaginal infections, ear infections, throat infections), eczema, allergies, lactose intolerance, high blood pressure, and/or heart diseases in a subject , comprising administering a therapeutically effective amount of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby or a composition comprising Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described elsewhere herein to the subject.

The term“therapeutically effective amount” as used herein, refers to an amount of active ingredient or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the active ingredients as taught herein.

In related aspects, the invention provides the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby or a composition comprising Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described herein for the manufacture of a medicament for the treatment of gastrointestinal diseases, auto-immune diseases (e.g., rheumatoid arthritis), infections (e.g. vaginal infections, ear infections, throat infections), eczema, allergies, lactose intolerance, high blood pressure, and/or heart diseases in a subject. In certain embodiments, Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby or a composition comprising Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as described herein is to be administered orally.

In certain embodiments of the methods and uses of the present invention, the animals, particularly arthropods, are provided with the Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, as described herein, ad libitum. The Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, optionally comprised within a composition as elsewhere described herein, can be placed close to (e.g. inside or just outside of) the domicile, the nest or the housing of the animal, particularly arthropod, or a natural feeding source of the animal and freely accessible to the animal. The domicile, the nest or the housing can be either natural or artificial and where the animal is a group-forming animal, the domicile, the nest or the housing typically houses several generations of animals.

As will be understood by the skilled person, the specific embodiments described for certain methods and uses as described herein are in most cases similarly applicable to all methods and uses as described herein. Furthermore, when referring to the use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby in the methods and uses as described herein, this also encompasses food compositions comprising Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, as described elsewhere herein.

An easy and preferred means for providing an animal with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as envisaged herein is via a food or feed composition comprising said Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as envisaged herein.

Accordingly, a further aspect provides food or feed compositions for animals comprising Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby. In particular embodiments, the Metschnikowia gruessii yeast is the Metschnikowia gruessii yeast strain with accession number 57395 (BCCM/MUCL culture collection), as elsewhere described herein.

The term“food composition” or“feed composition”, as used herein generally refers to a combination of elements which can be ingested by an organism without causing harm to the organism. Preferably, at least a part of said elements are essential and/or non- essential nutrients which can be ingested and assimilated by an organism to produce energy, stimulate growth and/or maintain life. Even more preferably, said food or feed composition is artificially produced. Feed composition typically denotes a food composition for non-human animals. In particular embodiments, the food composition is a non-naturally occurring food composition.

In certain embodiments, the food composition as envisaged herein is a dietary supplement. The term“dietary supplement” as used herein refers to a product intended to supplement or complement a subject’s diet, the product comprising one or more substances with a nutritional and/or physiological effect on a subject. The dietary supplement can be a partial nutritional composition, which does not contain all the essential macro- and micronutrients and hence may not be used to replace one or more daily meals and/or may not be used as the sole source of nutrition of a subject.

In particular embodiments, the food composition as described herein may be a liquid preparation, a powder, a gel, a paste, a tablet (e.g. a swallowable tablet, a chewable tablet), a concentrate, a suspension or a ready-to-use formulation.

In particular embodiments, the food composition as described herein is configured for oral administration.

The recitation“configured for oral administration” as used herein refers to the capability of the food composition to be administered through the oral cavity of a subject to the digestive system. Oral delivery of the food composition allows the ingredients of the dietary supplements to enter the gastrointestinal tract of the subject.

The skilled person will understand that the form of the food composition as described herein is dependent on the type of animal to which the food composition is provided or administered. For example, if the animal is a pollinating insect, the food composition is preferably a sugar solution or pollen, while if the animal is a human, the food composition is preferably a soluble powder, a solid tablet, or a drinkable liquid.

In particular embodiments, the food composition as described herein is a liquid food composition, preferably the food composition as described herein is an aqueous food composition.

In particular embodiments, the food composition as described herein comprises at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 2500, at least 5000, at least 10000, at least 25000, or at least 50000 of Metschnikowia gruessii yeast cells per milligram or per microliter of the food composition or the equivalent thereof in fragments or products derived therefrom. Preferably, the food composition as described herein comprises at least 100 Metschnikowia gruessii yeast cells per milligram or per microliter of the food composition or the equivalent thereof in fragments or products derived therefrom.

In certain embodiments, the food composition as described herein comprises between 10² and 10⁸, between 10³ and 10⁷, between 10⁴ and 10⁶, preferably between 10⁴ and 10⁵ of Metschnikowia gruessii yeast cells per milligram or per microliter of the food composition.

The Metschnikowia gruessii yeast cells may be present in the food composition in an active (i.e. alive) or inactive (death) form, or a combination thereof. Preferably, the Metschnikowia gruessii yeast cells are present in the food composition in an active (i.e. alive) form. Methods for inactivating or fragmenting yeast cells are known in the art. Non-limiting examples include heat shock, autoclaving, filtering, irradiation (gamma, UV).

In particular embodiments, the number of yeast cells in the compositions increases naturally over the time period that the composition is offered to the arthropod. In particular embodiments, the methods of the present invention involve offering the food composition as described herein to the arthropod for a period of between 1 and 30 days, such as for between 5 and 20, such as for 7 days. Within this time period, the concentration of active yeast cells in the food composition may increase. For instance, in particular embodiments, the food composition comprises 100 yeast cells/microliter, and increases up to 60000 cells per microliter in 7 days.

Metschnikowia gruessii yeast cells can be propagated according to any method for propagating yeasts known by the skilled person before adding it to the food composition as described herein. For example, yeast cells can be grown in a liquid growth medium, such as Yeast Malt broth, preferably comprising Peptone, yeast extract, malt extract, glucose and agar, at about 24°C for 24 to 48 hours standing on a shaking platform (e.g. 80-100 rpm). The concentration of yeast cells in a liquid medium, such as a liquid growth medium, can be measured using standard techniques, such as calculating the optical density of solution containing the yeast cells using a spectrophotometer or conducting cells counts of stained cells (the use of Methylene blue would allow to discern their viability, for instance) in an haemocytometer under the microscope at 40x magnification. Alternatively, the optical density may be measured with a refractometer at 600 nm. In certain embodiments, the food composition as described herein further comprises at least one carbohydrate source and/or at least one nitrogen source.

The term“carbohydrate” or“saccharide” as used herein refers to a biological molecule comprising carbon, hydrogen and oxygen. Non-limiting examples of carbohydrates are monosaccharides, such as fructose, glucose and galactose, disaccharides such as sucrose, lactose and maltose, oligosaccharides such as maltodextrin, raffinose and stachyose, and polysaccharides, such as starch, glycogen and cellulose. Carbohydrates form an essential part of the diet of many animals. Carbohydrates are mainly used to generate energy for muscular activity, body heat, and vital functions of certain organs and glands. Furthermore, carbohydrates, such as sugar, can act as feeding stimulants for animals, such as arthropods. Additionally or alternatively, carbohydrates can function as a growth substrate for the yeast.

In certain embodiments, the food composition as described herein comprises at least 10 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt%, at least 50 wt% or at least 60 wt%, preferably at least 30 wt% of one or more carbohydrates, preferably wherein said carbohydrate is sugar.

In certain embodiments, the food composition as described herein further comprises from 10 wt% to 70 wt%, from 10 wt% to 60 wt%, from 20 wt% to 50 wt%, preferably from 20 wt% to 40 wt% of one or more carbohydrates, preferably wherein said carbohydrate is sugar.

The term “sugar” as used herein refers to soluble carbohydrates. Non-limiting examples of sugars comprise glucose, fructose, galactose, sucrose, maltose, lactose, galactose, mannose, raffinose, dextrin, inulin, rhamnose, xylose, arabinose, trehalose, sorbose, ribose and melezitose. Non-limiting examples of sources of glucose, fructose, galactose, sucrose, maltose, lactose, galactose, mannose, raffinose, dextrin, inulin, rhamnose, xylose, arabinose, trehalose or melezitose are molasses, sugar beet sugar, cane sugar and hydrolysed starches.

In particular embodiments, said carbohydrate source comprises one or more of sucrose, glucose, maltose, dextrose, fructose, invert sugar, corn syrup and/or glucose syrup. In more particular embodiments, said carbohydrate source comprises one or more sugars selected from the list comprising sucrose, maltose, glucose, fructose and dextrose.

In particular embodiments, total amount of sugar present in the food composition as described herein comprises from 20 to 70wt%, preferably from 50 to 70 wt%, of sucrose; from 5 to 50 wt%, preferably from 10 to 20 wt%, of glucose; and from 5 to 50 wt%, preferably from 10 to 20 wt%, of fructose.

In particular embodiments, the total amount of sugar present in the food composition as described herein comprises from 50 to 70 wt% of sucrose, from 10 to 20 wt% of glucose and from 10 to 20 wt% of fructose (based on dry weight). In more particular embodiments, the sugar in the sugar solution consists of 66 wt% of sucrose, 16.6 wt% of glucose and 16.6 wt% of fructose (based on dry weight). Preferably, the sugar solution is sugar water having a concentration of 30 wt% of sugar, wherein the sugar is comprises from 50 to 70 wt% of sucrose, from 10 to 20 wt% of glucose and from 10 to 20 wt% of fructose (based on dry weight).

In particular embodiments, the carbohydrate source is sugar, nectar, honey or a substitute thereof.

It is noted that the food composition as described herein may additionally or alternatively comprise carbohydrates which are secreted by the Metschnikowia gruessii yeast cells, such as, but not limited to, neo-kestose, 6-kestose, bifurcose, and mannotriose.

In particular embodiments, a sugar alcohol, which is a modified form of a sugar, can be used instead of sugar. Non-limiting examples of sugar alcohols include glycerol, glucitol, sorbitol and mannitol.

In particular embodiments, the food composition as envisaged herein is a sugar solution, preferably a sugar solution comprising at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt% or at least 30 wt% of sugar. In particular embodiments, the food composition as envisaged herein is a sugar solution comprising at least 10 wt% or at least 20 wt%, more preferably at least 20 wt%, of sugar.

In particular embodiments, the sugar solution as envisaged herein comprises from 5 wt% to 50 wt%, from 5 wt% to 40 wt%, from 10 wt% to 30 wt%, from 10 wt% to 40 wt%, from 10 wt% to 50 wt%, from 20 wt% to 50 wt%, from 25 wt% to 50 wt%, from 30 wt% to 50 wt%, from 20 wt% to 60 wt%, from 25 wt% to 60 wt%, from 30 wt% to 60 wt%, from 20 wt% to 70 wt%, from 25 wt% to 70 wt%, or from 30 wt% to 70 wt%, preferably from 10 wt% to 30 wt% of sugar.

The sugar can be solubilised in, for example, water, milk or fruit juice. Preferably, the sugar is solubilized in water. Accordingly, in particular embodiments, the sugar solution is sugar water. The amount of sugar present in the food composition can be measured by methods known in the art. Non-limiting examples of such methods include high-performance liquid chromatography (HPLC) and a refractometer to detect sucrose equivalent in Brix. In certain embodiments, at least 80 wt%, at least 85 wt%, or at least 90 wt%, preferably at least 95 wt%, for example 97 wt%, 98 wt%, 99 wt% or 100 wt%, of the total amount of nutrients present in said food composition as described herein, is a sugar or sugar alcohol.

In particular embodiments, the nitrogen source is pollen or suitable substitutes thereof, amino acids, peptides, polypeptides, and/or proteins, preferably pollen. Pollen can be either naturally occurring pollen (e.g. natural bee pollen), or a synthetic pollen (e.g. synthetic bee pollen).

In particular embodiments, the food composition as described herein is pollen (e.g. a pollen ball) prepared with a sugar solution (e.g. sugar water), wherein said sugar solution comprises Metschnikowia gruessii, fragments thereof or substances produced thereby. In particular embodiments, the pollen comprises from 5 wt% to 20 wt%, from 5 wt% to 15 wt%, from 8 wt% to 12 wt%, or from 5 wt% to 10 wt% of a sugar solution (e.g. sugar water) comprising Metschnikowia gruessii. Preferably, the pollen comprises about 10 wt% of a sugar solution (e.g. sugar water) comprising Metschnikowia gruessii. In further particular embodiments, the pollen comprises a sugar solution (e.g. sugar water) comprising at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1000, at least about 2500, at least about 5000, at least about 10000, at least about 25000, or at least about 50000 Metschnikowia gruessii yeast cells per microliter or the equivalent thereof in fragments or products derived therefrom.

In particular embodiments, the pollen comprises at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt% or at least 30 wt% of protein. The protein can be present in the pollen in the form of, for instance, soy flower, torula yeast or brewer’s yeast. Methods for preparing pollen balls are known in the art.

In particular embodiments, the food composition as envisaged herein comprises one or more of a carbohydrate source, a nitrogen source, vitamins, lipids, fats, and/or minerals.

The term “lipid” as used herein refers to a substance that is soluble in nonpolar solvents. Non-limiting examples of lipids are fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipis and polyketides. The term“fatty acid” as used herein refers to a carboxylic acid with an aliphatic chain comprising at least 4 carbon atoms (e.g., from 4 to 28 carbon atoms). The fatty acid may either comprise carbon-carbon double bonds, and accordingly may be referred to as“unsaturated fatty acid”, such as monounsaturated fat, polyunsaturated fat and trans fat, including cholesterol. If the fatty acid does not comprise carbon-carbon double bonds, it may be referred to as a“saturated fatty acid”. Fatty acids may be present in a subject’s diet in the form of animal or vegetable fats and/or oils, such as lard, butter, milk fat, coconut oil, cocoa butter, palm kernel oil, soybean oil, corn oil, sunflower oil, hemp oil, rapeseed oil, wheat germ oil and cottonseed oil.

The term“vitamin” as used herein refers to an organic compound which is essential for normal growth and/or nutrition of a subject. Vitamins are typically required in small quantities in the diet of the subject or as a supplement to the diet. Non-limiting examples of vitamins include vitamin A, vitamin B1 , vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E and vitamin K.

The term“mineral” as used herein refers to an edible metal or a salt thereof such as zinc or a salt thereof, selenium or a salt thereof, tin or a salt thereof, and copper or a salt thereof.

In certain embodiments, the food composition as described herein may further comprise one or more pharmaceutical excipients. Suitable pharmaceutical excipients depend on the dosage form and identities of the active ingredients and can be selected by the skilled person (e.g. by reference to the Handbook of Pharmaceutical Excipients 6th Edition 2009, eds. Rowe et al.). As used herein,“carrier” or“excipient” includes any and all solvents, diluents, buffers (such as neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as EDTA or glutathione), amino acids (such as glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilisers, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Such materials should be non-toxic and should not interfere with the activity of the active ingredients.

In certain embodiments, the food composition as described herein may meet the general nutritional requirements of arthropods. Accordingly, the food composition as described herein may be used to provide arthropods with a fully nutritious, easily digestible, complex mixture of nutrients in amounts and proportions effective to support growth, development, maintenance, and reproduction. Thus, in particular embodiments, the food composition as described herein is specifically adapted to use as feed for arthropods.

In certain embodiments, the food composition as envisaged herein may comprise other organisms, such as bacteria and/or yeast, in addition to Metschnikowia gruessii yeast cells. In certain embodiments, Metschnikowia gruessii yeast is the main organism present in the food composition as described herein, meaning that Metschnikowia gruessii yeast is present in the largest numbers in the food composition as described herein. In preferred embodiments, the food composition as described herein does not comprise any organisms, fragments thereof and/or substances produced thereby in addition to Metschnikowia gruessii yeast, fragments thereof and/or substances produced thereby.

In certain embodiments, Metschnikowia gruessii yeast, fragments thereof and/or substances produced thereby is the sole active ingredient present in the food composition as envisaged herein.

The terms“active ingredient” or“active component” can be used interchangeably and broadly refer to a compound or substance which, when provided in an effective amount, achieves a desired therapeutic and/or prophylactic outcome(s). Typically, an active ingredient may achieve such outcome(s) through interacting with and/or modulating living cells or organisms. The term “active” in the recitations “active ingredient” or“active component” refers to“pharmacologically active” and/or“physically active”.

In particular embodiments, the food composition as envisaged herein may be sterilised or pasteurised to increase the shelf life of the food composition. Preferably, the sterilisation or pasteurisation of the food composition as described herein is performed prior to the addition of the Metschnikowia gruessii yeast cells thereto. Where the food composition is not envisaged to comprise live Metschnikowia gruessii yeast cells, but rather comprises inactivated yeast cells, yeast fragments or products derived from yeast cells, sterilisation or pasteurisation of the food composition as envisaged may be performed after the addition of the yeast cells, yeast fragments or products derived from yeast cells to the food composition. The sterilisation or pasteurisation of the food composition as described herein may be performed by any methods known in the art to sterilise or pasteurize food compositions. For example, it may be performed by ultraviolet germicidal irradiation. Alternatively or in addition, the food composition may comprise a suitable preservative and/or antimicrobial agent for preventing spoilage of the food composition and/or to enhance its shelf life. Suitable preservatives or antimicrobial agents are known in the art and are preferably not harmful for the Metschnikowia gruessii yeast cells comprised within the food composition.

In certain embodiments, the food composition as described herein may be stable for at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, or at least 42 months, preferably when stored at room temperature or at a temperature of 4°C.

The application further provides methods for preparing a food composition as described herein, the method comprising the step of preparing a mixture of Metschnikowia gruessii yeast cells, fragments thereof and/or substances produced thereby, and at least one ingredient selected from a carbohydrate source or a nitrogen source; thereby obtaining the food composition.

The ingredients of the food composition as envisaged herein can be mixed according to standard methods known by the skilled person.

In particular embodiments, the method for preparing a food composition as described herein comprises the step of preparing a mixture of Metschnikowia gruessii yeast cells, fragments thereof and/or substances produced thereby; at least one ingredient selected from a carbohydrate source and a nitrogen source; and one or more lipids, vitamins or minerals.

In certain embodiments, the mixture is a homogeneous mixture.

In particular embodiments, the method for preparing a food composition as described herein comprises inoculating a sugar solution (e.g. sugar water) as described herein with Metschnikowia gruessii yeast cells. In particular embodiments, the methods the method for preparing a food composition as described herein comprise mixing pollen with a sugar solution (e.g. sugar water) comprising Metschnikowia gruessii yeast cells so as to obtain a concentration of about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1000, at least about 2500, at least about 5000, at least about 10000, at least about 25000, or at least about 50000 Metschnikowia gruessii yeast cells per microliter or the equivalent thereof in fragments or products produced thereby. More particularly, this can encompass inoculating a concentrated yeast inoculum in a sugar solution (e.g. sugar water). In particular embodiments, the sugar solution comprises from 5 wt% to 50 wt%, from 5 wt% to 40 wt%, from 10 wt% to 30 wt%, from 10 wt% to 40 wt%, from 10 wt% to 50 wt%, or from 20 wt%, preferably from 10 wt% to 30 wt%,of sugar. In particular embodiments, the methods further comprise mixing the sugar solution (e.g. sugar water) with pollen so as to obtain a pollen ball. Further embodiments of the methods are related to the food compositions as described herein.

In certain embodiments, the food composition as envisaged herein can be provided to arthropods by any form of feeding device for arthropods known by the skilled in the art. Advantageously, a liquid food composition can be placed in a container and/or can be applied onto porous or fibrous items, such as a cotton ball or a capillary wick, which can be placed close to the nest or in the nest box and is available to the arthropod via capillary action. When the arthropods are pollinating insects, the food composition according to the invention can also be provided or sprayed on flowering crops. When the arthropods are pollinating insects, the food composition according to the invention can also be provided by artificial flowers comprising an electromechanically operated dispensing device. Preferably, the food composition is substantially odor-free, or if such food composition contains an odor, such odor should not be malodorous or repellent to arthropods, especially to bees.

In particular embodiments, the food composition comprises volatiles (e.g. alcohols, aldehydes, esters) produced by Metschnikowia gruessii yeast cells which may attract animals, more particularly athropods.

In particular embodiments, the presence of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby in a food source may increase the intake of said food source, as Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby may act as phagostimulants.

Further, the inventors have isolated a specific Metschnikowia gruessii yeast strain from a pollenpot of a Bombus terrestris colony. Advantageously, the provision of this specific Metschnikowia gruessii yeast strain as envisaged herein to arthropods, more particularly bees, even more particularly B. terrestris, had a pronounced positive effect on the rate of development of workers, the number of workers, the female fitness, the brood per nest and/or the size and weight of workers. These beneficial effects were absent in arthropods not receiving any Metschnikowia gruessii yeast cells.

Accordingly, further provided herein is a specific strain of Metschnikowia gruessii yeast, more particularly the Metschnikowia gruessii yeast strain deposited by Jean-Marc Vandoorne-Feys in the name of Biobest Group on December 5, 2018 in the BCCM/MUCL (Belgian Coordinated Collections of Micro-organisms (BCCM), Universite catholique de Louvain; Mycotheque de I’Universite catholique de Louvain (MUCL), Croix du Sud2, box L7.05.06, 1348 Louvain-la-Neuve, Belgium) under the accession number 57395 (see Table A) or variants or derivatives thereof. Said strain or variants or derivatives thereof can be used to advantageously rear animals, preferably arthropods (e.g. pollinating insects), and/or to improve the health, behaviour and/or general fitness of said animals, as elsewhere described herein. Table A. Indications relating to deposited microorganism 57395

As referred to herein, the term “variants” refers to microbial variants such as mutational, insertional, and deletional variants of Metschnikowia gruessii yeast strain 57395 as well as microbial variants having a whole genome sequence identity of at least 90%, more preferably at least 95% and for instance at least 96%, 97%, 98%, 99% or 99.9%.

The following examples are provided for the purpose of illustrating the present invention and by no means are meant and in no way should be interpreted to limit the scope of the present invention. EXAMPLES

Example 1 : The use of Metschnikowia gruessii yeast cells to optimize arthropod rearing: sugar water modification

1.1 Bumblebees

B. terrestris colony development was followed-up over a 16-week-period. A total of 60 colonies were kept under standard climatic conditions (28°C and 60% relative humidity) and fed ad libitum with sterilized honeybee-collected pollen and a 50% sugar water solution (wherein the sugar consisted of 2/3 Sucrose (S), 1/6 Fructose (F), 1/6 Glucose (G)). Next to this diet, control colonies (N=12) received 30% sugar water (same composition of sugar as the aforementioned 50%) plus sterile Yeast Malt Broth (YMB) in a proportion of 50 pi per each milliliter of sugar water. The remaining colonies (N=48) also received a second sugar water feeder, with composition as listed in Table 1. All sugar solutions were free of preserving agents, they were refreshed weekly and administered via two separate containers underneath the brood box.

Table 1. Experimental design for symbiont administration via sugar water

Abbreviations: F, fructose; G, glycose; OD, optical density; S, sucrose; YMB, Yeast Malt Broth

^* the number of living yeast cells will increase with time, but will be less than 1000 cells/mI after one week

The larval developmental time was assessed by tracking the timing of first egg-laying, first development into pupae and first emergence of adults. The number of adult workers, pupae, larvae, dead larvae, and eggs was counted at week 5 and week 10. At week 1 1 , 5 fully developed colonies (colonies with more than 80 workers) were selected per treatment and transferred into bigger boxes where they were allowed to develop further another 5 weeks after transfer. After this, the trial was finalized and all aforementioned parameters were assessed with one additional parameter being the total number of sexuals (males and queens), as this is a parameter for colony fitness. The sum of pupae and workers, hereafter named as“predicted workers” was used as an additional variable to assess colony performance. A higher number of predicted workers at a certain point in time for treatments 1 , 2, 3 and/or 4 compared to the control treatment indicates that colonies fed with M. gruessii develop faster. A more rapid colony development could imply a higher egg laying rate, a faster larval development and/or a higher larval survival. The administration of M. gruessii is demonstrated to have at least two of these effects. First, the timing to produce workers is up to a week faster when M. gruessii was administered (Figure 1 ). The results of counts after a 5 and a 9-week-developmental period show that there is a significant increase, namely a 2-fold increase, in the number of workers when compared to control colonies that did not receive the M. gruessii yeast treatment, regardless of the way of administration (Figure 2).

Present inventors showed that the beneficial effect of feeding M. gruessii to insects can be obtained by different, non-exclusive, pathways: administering the live M. gruessii yeast (treatment 1 ), the medium modified by the M. gruessii yeast with the cells present (treatment 2) and the inactivated M. gruessii yeast cells only (treatment 3). All these treatments had a similar positive effect on colony development, resulting in a faster development with higher number of workers produced compared to the control treatment (Figure 2). A comparison of early development at week 5 with fully developed nests after 9 weeks, allowed to better corroborate that differences between treatment 1 and 3 reached statistical significance (t = -4.8, p= 0.0004). More importantly, differences in respect of the control treatment were always significant (t= -15.3, -19.29, -19.42 for paired comparisons of control treatment with treatment 1 , 2, and 3, respectively, p < 0.0001 ).

Administration of M. gruessii also had a significant positive effect on the female fitness after a 16-week-period (Figure 3). Besides, female fitness differences in respect of the control treatment were mostly driven by differences in number of workers, and not in queens, which would affect positively the sellable stage of the hives and their flight activity on the field. In this regard, the amount of queens produced was not different along control, treatment 1 and treatment 3. Only treatment 2 lead to slightly higher count of queens (results not shown). One important aspect that affects the saleability of bumblebee hives for pollination, and their foraging activity in the field, is the number of males produced. In this regard, substantial differences were obtained at the number of males produced by comparing all forms of M. gruessii administration with respects to the control treatment. Still, the effect was even more significant when this M. gruessii was applied in the alive form to the bumblebee liquid diet (Treatment 1 , Figure 3).

It was confirmed that colonies consumed the container with treatment diet every week, and for treatment 2 the first sugar water feeder comprising M. gruessii was strongly preferred over the second sugar water feeder containing the standard 50% sugar water diet (Figure 4). The administration of M. gruessii not only led to the production of much more workers per nest, the size and weight of those individual specimens tended also to be bigger (Figure 5).

1.2. Solitary bees: Osmia bicornis

The effect of nectar-living yeasts on the fitness of a second insect, namely the solitary bee Osmia bicornis, was studied. A total of 20 O. bicornis pupae (50% females, the remaining half males) were harvested in the wild and then split into 4 bug dorm cages of 60 cm x 60 cm, by keeping 5 female pupae and 5 male pupae per nest. Each treatment was applied in two separate cages. The pupae were put in a wooden bee hotel, and were subsequently offered 2 containers with sugar water, one with sterile 50% sugar water and a second with M. gruessii treatment 1 (as described Table 1 ). Bees had access to pollen feeders (multifloral -honeybee collected-, apple, and pine), water, and sterile soil in separate petri dishes. Treatments were refreshed weekly. During a period of 2 weeks the fitness of each treatment was monitored by tracking survival rate.

Example 2. The use of M. gruessii to optimize arthropod rearing: pollen -“bee bread”- modification

The yeast M. gruessii strain 397 (57395) have been isolated from a pollenpot of a commercial B. terrestris colony that was foraging freely in the wild for 4 weeks. From a survey within 8 colonies that were placed in the wild, just 50% of them built pollenpots. M. gruessii strain 397 was isolated from one pollenpot of one of this colonies. The whole genome of this strain has been sequenced for further characterization of the strain.

Given the origin of the isolate, M. gruessii strain 397 was added to the pollen-sugar water mix that the bumblebees receive in the mass rearing on a weekly basis. Bee bread was prepared by mixing 80% of honeybee-collected pollenpelets with up to 20% of 40% sugar water (the sugar consisting of 1/3 Sucrose, 1/6 Fructose, and 1/6 Glucose) free of preserving agents. In the M. gruessii treatment, the strain 397 was suspended in YM broth, until an OD of 0.5 was achieved at the day of food preparation. At control treatment, no yeasts are added to YM. The colonies receive ad libitum Biogluc® underneath as sugar water source. A helper worker (white worker) was added 4 days after placing the queen, and first eggs were laid around 8-10 days after queen start-up. First workers appear after 35 days, on average, for the control treatment, and this appearance occurred the fastest for queens treated with symbionts. Consistent with the temporal overview, the first counting, performed at week 5, revealed that a significantly larger brood was achieved by queens treated with M. gruessii (results not shown). At 8 weeks, before the colonies had limitations in space, the trial was finished by freezing the colonies. Then, all elements of individual nests were counted. It was found that colonies treated with M. gruessii strain 397 in the pollen had a significant larger brood and a significantly larger amount of workers (Figure 7).

Example 3. The use of Metschnikowia gruessii yeast cells to alter the behavior of arthropods

3.1. Behavior

A translation of the results from examples 1 and 2 was made to agricultural practices, by testing the attractiveness of any of the M. gruessii treatments (yeasts alive, inactivated, solution filtered, solution plus cells) to individual B. terrestris workers. This is achieved by placing robotic flowers on an experimental arena, in which a fully developed bumblebee colony is placed. Colonies were never in contact with the treatment diet and never foraged before.

In order to test reference, a series of 2-choice experiments was developed. The number of flower visits to each treatment plus the probing time is taken as attractiveness measures.

A behavioral test has been done to compare the attractiveness of M. gruessii treatment when added to artificial nectar, compared to control conditions in which no yeasts where present. Both the control treatment and the M. gruessii treatment 1 were prepared as described in Table 1. One indoor-reared B. terrestris colony of 10 weeks, that was not in contact with microbes in the food before, was chosen. One day before the starting of each trial, the Biogluc® access of the hive was turned off to starve the bees and prepare them to forage. The colony was then open inside a greenhouse equipped with an experimental flying arena of 2.5m x 3 m. This arena contained 16 robotic flowers, plus a yellow plastic ring as landing platform, divided in 4 groups. This system mimics the system described by Kuusela et al (“A low-cost, computer-controlled robotic flower system for behavioral experiments”, Ecol Evol, 6(8), April 2016). The system includes a control unit, separate flowers, and a personal computer. The function of the control unit is to handle the electronics of the flowers, collect data from them, and to send data to the computer. The flowers themselves contain an infrared (IR) sensor, their associated electronics, and an electromechanical device (servo) that offers a small, precise amount of sugar solution (referred from hereafter as“nectar”) from a reservoir. The computer runs software that controls the refilling rate and data collection via the control unit. The rationale of the system is that the entrance of the bumblebees in the artificial flowers causes a voltage drop that is registered by a custom-made Java interface. Date and time of visitation, as well as flower identity, is registered for each visit, as well as probing time for each effective visit. Flower depletion in wild flowers is mimicked by setting an automatic refilling period of 10 minutes. In such case, sugar water is not continuously offered and visitation rate approaches probing time in wild flowers.

The trial was composed of 16 artificial flowers with half of the flowers containing the control and half the sub treatment. Along 4 days, the number of effective visits to flowers with control treatment (without yeasts) and M. gruessii treatment 1 , as well as the total time spent consuming nectar from each flower type, were recorded.

It was shown that naive bumblebees preferred flowers that contained alive M. gruessii, as those visits last significantly longer (Figure 8).

Example 4: Use of Metschnikowia gruessii yeast cells in arthropod rearing

The effect of M. gruessii strain (up to 3 different strains), cell density (up to 3 dosages) and way of administration (by pollen or sugar water) on arthropod rearing was evaluated.

4.1 Experimental setup

B. terrestris queens were fed ad libitum with a 50% sugar water solution (wherein the sugar consisted of 2/3 Sucrose (S), 1/6 Fructose (F), 1/6 Glucose (G)). Half of the queens received in addition pollen supplementation; the other half had access to a second sugar water feeder, both of which were refreshed weekly. All sugar solutions were free of preserving agents, they were refreshed weekly and administered via two separate containers underneath the brood box. For both forms of administration, the following treatment combinations were evaluated (N=15 for each treatment):

- Control - no micro-organism - M. gruessii strain 397 (BCCM/MUCL number 57395): 25 (3); 50 (2) and 100 (1 ) cells per mI;

- M. gruessii strain 423 (from tongue): 25 (3); 50 (2) and 100 (1 ) cells per mI;

- M. gruessii strain 453 (from gut): 25 (3); 50 (2) and 100 (1 ) cells per mI.

All treatments further comprised Yeast Malt Broth (YMB) in a proportion of 50 mI per each milliliter of sugar water. For each treatment, the date of first egg and first worker was determined. Colony size was determined at week 5 and week 10.

Field evaluation At week 10, a subset of the colonies was further evaluated in the field, i.e. the colonies fed with sugar water comprising 100 cells of the micro-organism per mI (N= 3 ^* 4) were further evaluated in the field. A total of 5 workers (week 0 + week 1 after placement in the field and 3 workers (week 2-4 after placement in the field) will be collected per hive at 5 consecutive weeks (before placement, week 0, and then 4 weeks on). The workers were dissected aseptically and the gut was collected. DNA was extracted from the gut content and the pathogen load was by qPCR using the protocol by Bosmans et al (2018). Hive activity was monitored as well, in particular the number of workers entering the hive with or without pollen and the number of workers exiting the hive, by performing 5 min censuses at least once a week with a minimum of 3 measurements per day.

Gut plating. 3 workers collected from different colonies were dissected aseptically and the gut content was plated onto YGC plates, including a 10x and 100x dilution of the sample. Colonies were identified by Sanger sequencing.

4.2 Results

Hive activity. The different M. gruessii strains had generally little impact on hive activity (i.e. the total number of incoming and exiting bumblebees per 5 min census). M. gruessii strain 397 (BCCM/MUCL number 57395) (at a dosage of 100 cells per mI) showed a small but not statistically significant increase in hive activity compared to the control treatment or to the other M. gruessii strains.

Impact on pathogen load (by qPCR). Pathogen load was evaluated by qPCR using the protocol by Bosmans et al (2018). Bumblebees which received M. gruessii strain 397 (BCCM/MUCL number 57395) as part of their diet had the fewest DNA copies of Crithidia bombi in the gut (about 10⁹ copies after 5 weeks). In comparison, bumblebees which were fed with the control diet without M. gruessii contained the largest number of DNA copies of Crihtidia bombi (about 10¹¹ copies). The C. bombi load was also reduced for the bumblebees fed with M. gruessii strain 423 (to about 1.3 x 10⁹ DNA copies after 5 weeks) or with M. gruessii strain 453 (to about 1.2 x 10¹° DNA copies after 5 weeks).

Impact on colony development: workers

Impact of the different treatments on the average numbers of workers per colony is shown in Figure 9.

There is a triple interaction among treatment (M. gruessii strain), way of administration (via pollen or sugar water) and dosage (A2 = 60.33, df = 4, P < 0.0001 ). Regarding the effect of treatment, all strains yield higher counts than the control (Z = 10.60, 10.98, 10.36, P < 0.0001 , for Strains 1 , 2 and 3, respectively). For strains 1 and 2, worker counts were significantly higher at a dosage of 100 cells per microliter in the case of pollen administration, while opposite trends were obtained for the sugar water administration, where a dosage of 25 cells per microliter lead the best results. Strain 3 has very consistent worker counts at week 10 regardless of the dosage and way of administration.

Impact on colony fitness: sexuals

Sexuals, either males or queens, did not appear earlier during the colony development, regardless of the differences that were spotted at week 10 for worker production among treatments. Overall, we can conclude that competition point did not occurr earlier for those colonies supplemented with yeasts. That would ensure bigger nests that might result in better visitation rates and pollination of the target crops without compromising their lifespan in the field.

For the sugar water administration, queens first appeared after ten weeks of development for one colony of the Strain 3 at the lowest dosage within the sugar water administration. The remaining colonies started having queens 2 weeks later. Nests corresponding to the pollen administration produced queens at week 1 1 at the earliest in control conditions and 9 days later for those supplemented with M. gruessii strains. Queen production at week 16 for colonies supplemented with the yeast M. gruessii were consistently lower or equal to control, even though colonies developed faster regarding the appearance of workers. First males appeared at week 5 irrespectively of the treatment for the pollen administration. For the pollen administration, males were first spotted at week 7 and again there were no differences among treatments. Number of males after 16 weeks of development followed the same trend that we previously described for workers at week 10.

In conclusion, supplying M. gruessii strains to bumblebees had a positive influence on bumblebee fitness (pathogen load in the gut) and colony development compared to a control diet without M. gruessii. M. gruessii strain 397 (BCCM/MUCL number 57395) gave the best results of the M. gruessii strains tested (for these parameters).

Claims

1. A method for rearing animals and/or improving the fitness of animals, comprising the step of feeding said animals with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby.

2. The method according to claim 1 , wherein said method is a method for improving the growth, the development, the reproductive capacity, the health and/or the behaviour of said animals.

3. The method according to claim 1 or 2, wherein said animals are arthropods.

4. The method according to any one of claims 1 to 3, wherein said animals are pollen- or nectar-consuming arthropods.

5. The method according to any one of claims 1 to 3, wherein said animals are pollinating arthropods.

6. The method according to any one of claims 1 to 5 wherein said animals are Hymenoptera, preferably bees or bumble bees.

7. The method according to any one of the preceding claims, wherein said animals are colony-forming arthropods and said method is a method for improving the colony development rate.

8. The method according to any one of the preceding claims, wherein said animals are pollinating and/or pollen- or nectar-consuming arthropods and/or flying arthropods and said method is a method for improving flight and/or foraging activity.

9. A method for cultivating a fruit-bearing crop, comprising the steps of:

- feeding one or more pollinating arthropods with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby; and

- ensuring pollination of the flowering crop by the one or more pollinating arthropods.

10. A method for cultivating a crop, comprising the steps of: - feeding one or more arthropods with Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby, wherein said one or more arthropod is a biological control agent for the biological control of pests; and

- contacting the one or more arthropod with the crop to ensure the biological control of pests on said crop.

1 1. A method according to any one of claims 1 to 10, wherein said Metschnikowia gruessii yeast cells are yeast cells from the Metschnikowia gruessii yeast strain deposited under the accession number 57395 at the BCCM/MUCL culture collection.

12. Use of Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby as a prebiotic and/or a probiotic, preferably for improving the immune system and/or immune function of animals, reducing the number of gut parasites, promoting the build-up of healthy gut bacteria and/or absorption of nutrients, support the digestive health and/or gut barrier function, inhibiting growth of harmful gut bacteria, and/or reducing stress.

13. Metschnikowia gruessii yeast cells, fragments thereof or substances produced thereby for use as a medicament, preferably for use in the prevention or treatment of gastrointestinal diseases, auto-immune diseases, infections, eczema, allergies, lactose intolerance, high blood pressure, and/or heart diseases.

14. A food composition for animals comprising at least 100 of Metschnikowia gruessii yeast cells per microliter or microgram of said food composition.

15. The food composition according to claim 14, further comprising one or more of a carbohydrate source, a nitrogen source, vitamins, lipids, fats, and/or minerals, preferably wherein said carbohydrate source comprises one or more sugars selected from the list comprising sucrose, maltose, glucose, fructose and dextrose.

16. A Metschnikowia gruessii yeast strain as deposited under the accession number 57395 at the BCCM/MUCL culture collection.