3
Introduction
4
Apis mellifera, overview of its biological cycle
The honeybee (Apis mellifera L.) is a worldwide studied organisms. It’s a very important organism for the agricultural economy, not only for the production of royal jelly, honey, bee- glue, but mainly for the pollination. In 2007, media attention focused on colony collapse disorder, a decline in European honeybee colonies in limited number of regions of North America.
Apis mellifera is a social insect that belongs to the order of the Hymenoptera and the Apidae family. The society of honeybees is matriarchal, monogynic, multi-year and is defined in two sexes and the females only are divided in two castes: queen and workers (Evans and Wheeler, 1999; Evans and Wheeler, 2000).
Honeybees show polyphenism (Rembold, 1976; Beetsma, 1979; Evans, 1999; Evans and Wheeler, 1999; Evans and Wheeler, 2000; Evans and Wheeler, 2001), that is the ability to develop into alternative morphologies with the same genome. The honeybee is an holometabolous insect. The queen is the only sexual reproductive female of the colony. The queen is fecundated by 4-5 drones in order to increase the genetic variability of the survivor of the colony. The egg is white and sub oval. The queen lays two kinds of eggs: haploid and diploid eggs. From the haploid eggs develop the drones which are sexual reproductive males. The queen will lay up to 2000 eggs/day for the next one to three years, until she dies or her sperm stores are depleted (Winston, 1987).
From the diploid eggs develop the female castes: workers and queens. The queen lays each egg in a cell prepared by the worker bees. The egg stage is 3-4 days long. The egg hatches into a small larva which is fed by 'nurse' bees (worker bees which maintain the interior of the colony).
Both larvae predestined to workers and to queens are fed royal jelly during the first three days of
the larval stage. Then workers are switched to a diet of pollen and nectar or diluted honey
5
(worker jelly) (H. Rembold, 1976), while queens will continue to receive royal jelly (J.D. Evans, 2001). This event is called “nutritional castration” and it is the basis of the phenotypic diversity between worker and queen and causes the regression of the reproductive apparatus of the worker (Pain, 1968; Contessi, 2007; Frediani and Pinzauti, 1993).
Fig. 1.1 Model of larval development of Apis mellifera.
After 5-6 days (Grandi, 1996) of larval stage there is the capping of the cells and begin the prepupae and pupae phases which have different length for drones, workers, or queens (Fig. 1.1).
The time of development from egg to newemerged queen is 16 days, for the worker is 21 days and for the drone is 24 days. With the impupament the larvae secretes a silk thread which is used to form the cocoon. The larvae predestined to become queen develop in the phase of pupae their 150-180 ovaries, while the larvae predestined to become worker, in the same phase, start to atrophy the ovaries and are left with 2-4 ovaries (Lin-Hua Rong et al., 1998).
In the queen, workers and drones (Fig. 1.2) the body is divided in three regions: the head, the
thorax and the abdomen. The head contains the antennae which have a sensory function, two
compound eyes and three simple eyes and the buccal system, formed by the ligule; in the thorax
there are two pairs of wings and three pairs of legs of which the third pair presents particular
6
small ”baskets” and a thick clump of bristles, adapted for the collection of the pollen; in the abdomen there are the gonads, only in the females there is the sting and only in the workers there are the beeswax glands. All the body is covered by chitin and hair. The workers are very complex because they change their anatomy depending on their task in the hive. There is a transition from working in the hive to foraging and this involves changes in the expression of thousands of genes. Denison and Raymond- Delpech examined three genes in particular, foraging, malvolio and vitellogenin, all implicated in this striking behavioural change in the life of the honeybee (Denison et al., 2008).
Fig. 1.2 The worker on the left, the queen in the middle and the drone on the right.
7
Honeybee larvae
The egg of the bees hatches in about 76 hours and the young larva curves itself in the opposite direction of the curvature of the egg and it will maintain this position during most of its larval life. From larva to adult occurs the metamorphosis, which may be defined as any change of form or structure that an animal undergoes which is not in line with direct development, or which results from a deviation from it (Snodgrass, 1925). The larva of the honeybees sheds its skin five times during the course of its growth. The larvae of diploid eggs are undifferentiated for 2 days and their food is a product secreted from nurse bees (6-12 days of image life), the royal jelly.
The dietary change within 2 days of eclosion determines whether a larva is destined to be a queen or a worker, due to epigenetic control (Elango et al., 2009). The anatomy of the larva (Fig.
1.3) is the same in all the 5 days but the size changes drastically. The most part of the space within the body cavity contains the mid gut. The hindgut and excretory tubules are not connected to the mid gut until the end of the larval development, when the insect stops to eat.
Fig. 1.3 Lateral dissection of the honeybees larva.
They have a white and relative soft exoskeleton and silk glands which begin to spin a cocoon in
the last half of the larval development. Levels of the immunity factors prophenoloxidase and
apismin are positively correlated with development suggesting a molecular explanation for the
8
reason why bees are susceptible to major age-associated bacterial infections such as American
foulbrood or chalkbrood (Chan et al., 2008). In the larval salivary gland of the honey bee there is
a pheromone that can cause effect, like the capping of the cells, the recognition of the larval age
and needs, effects stimulating hypopharyngeal glands of nurses and inhibiting ovary
development (Le Conte, 2006).
9
Glands responsible for the royal jelly production
The mandibular gland lies within the head of the worker just above each mandible, and has the form of a small bilobed sac opening with a narrow neck in the membrane at the inner side of the base of the mandible in front of the attachment of the flexor apodeme (Snodgrass, 1925). The compounds produced in the mandibular glands of A. mellifera workers are involved in food preservation and larval nutrition (Plettner et al., 1997).
The hypopharyngeal glands (Fig. 1.4) are a pair of long glands forming many loops and coils at both sides of the head. Each gland consists of numerous small oval cellular bodies attached by short necks to an axial duct. There are as many as 550 lobules on each duct (Snodgrass, 1956).
The hypopharingeal gland of the bees goes through morphological and physiological changes.
Morphologically their acini are well developed in the nurse worker bees (6-12 days image life) and begin to shrink in size after day 15 when a bee becomes a forager (Li et al., 2011).
Physiologically, there is higher activity in protein synthesis during the nursing period and lower in foraging bees (Li et al., 2011). Ohashi et al. (1997) showed that the individual secretory cells of an acinus of the hypopharyngeal gland express genes differently with the age-dependent role change of the worker bee. But the age at which the hypopharingeal glands degenerate is quite variable and depends on the colony conditions and the time of the year (Hrassnigg et al, 1998).
The function of the hypopharyngeal gland cells of the worker is flexible and can, if necessary, be maintained as that of the nurse, depending on the condition of the colony (Ohashi et al., 2000).
Brood provides the workers with a signal, which induces protein synthesis in the glands (Huang
et al., 1989). Costa and Cruz-Landim (2005) found fifteen different hydrolase enzymes in the
extracts of honey bees hypopharingeal glands (table 1.1).
10 Table 1.1 Enzymatic activity of hypopharingeal glands extracts from workers of honey bees as nmol of digest substrate. From Costa and Cruz-Landim (2005).
Huang and Otis (1989) showed that glands of 3 to 16 day old bees from broodless colonies have
a reduced synthesizing activity compared to glands of bees from brood right colonies. The
vitellogenin serves as a precursor to brood food proteins secreted by the hypopharingeal glands
of worker bees (Wegener et al., 2009). The secretion of the hypopharingeal gland and of the
mandibular gland is a sticky, milky fluid: the royal jelly (Dade, 1962; Townsend et al., 1940; Qu
et al., 2008). This white-yellow colloid has a pH of 3.6-4.2 and its composition is variable
depending on the metabolic and physiologic state of nurse bees, on the larval age, on the honey
bee race and seasonal and regional conditions (Scarselli et al., 2005). The royal jelly is usually
sampled from cells containing 72h old queen-designate larvae and contains water (60-70%),
proteins (12-15%), carbohydrates (10-12%), lipids (3-7%), and traces of mineral salts and
vitamins. The Major royal jelly proteins (today named apalbumins) constitute about 90% of the
11
total proteins of the royal jelly (Schmitzová et al., 1998; Albert et al., 2004). The royal jelly contains also antimicrobials peptides. Jelleines are a family of antimicrobials peptides in the royal jelly active against yeasts, Gram+ and Gram- bacteria (Fontana et al., 2004). Another antimicrobial peptide is the royalisin, active against Gram+ bacteria and fungi (Fujiwara et al., 1990). Recently the antimicrobial peptide defensin1 was found in the royal jelly and it is able to inhibit in vitro growth of the Paenibacillus larvae, the etiological agent of the American foulbrood (Klaudiny et al., 2012). The enzymes alpha glucosidase, glucose oxidase and alpha amylase are produced in the hypopharingeal glands of nurse bees and secreted into the royal jelly (Santos et al., 2005).
The post cerebral glands are located behind the brain. The acini are more translucent than the creamy bodies of the brood-food glands, have a different, characteristic shape and are arranged in small groups on a branching system of tubules (Dade, 1962). These acini go into a median duct which brings also the secretion of the salivary glands in the thorax. The honeybee’s salivary system might synthesize and secrete some enzymes that are involved in digesting nectar and/or pollen, or proteins/molecules that are secreted into the royal jelly, and which might affect the physiologic conditions of other colony members (Fujita et al., 2010).
Fig. 1.4 Schematic drawing of the food processing gland. From Dade, 1962.
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Circulatory system
In all the insects, bee blood, called hemolymph, is distributed throughout the body in an open circulatory system (Fig. 1.5). The blood of the adult honeybees is a pale, brownish liquid containing a particular kind of hemocytes. The blood of the larva is a clear liquid constituting from 25 to 30 per cent of the total weight of the larva. Though there are no special blood vessels such as veins and arteries in insects, there may be definite spaces separeted from the rest of the cavity by transverse sheets of membranous or muscle tissue known as the dorsal and ventral diaphragms (Snodgrass, 1925). The diaphragms contain muscle fibers which are rhythmically contractile so they give a direction to the circulation of the blood. In the dorsal sinus there is an organ, the heart or the dorsal vessel that is a median tube extending through most of the length of the body and that is independently contractile. It is the chief blood-propelling organ of the insect (Snodgrass 1925).
Fig. 1.5 Schematic drawing of the longitudinal section of the entire body of worker with heart and diaphragms.
(Dade, 1962)
The section of the circulatory system is important to study the modality of infection of some
parasites for example the infection with the Varroa mite.
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The immune system of Invertebrates
Immunity is a term that many would see reserved to the adaptive immune response on the basis of clonal expansion and antibody production as found in Vertebrates. However, recent progress in the study of Vertebrates (and plants) innate immunity uncovered deep homologies with the invertebrate immune defense, that suggest that this weapon of the immune system plays a crucial role as a first line of defense and in the organization of the subsequent adaptive responses (Brown P, 2001; Paul Schmid-Hempel, 2005). All evidence to date indicates that invertebrates (most data are from the arthropods) probably do not produce antibody (i.e., gamma globulins), and yet they possess a degree of natural immunity to certain pathogens and can be actively and passively immunized against pathogens (Taylor, 1969). As opposed to vertebrates, invertebrate responses whether local or systemic, do not involve a clonal amplification of the cells producing a given effector molecule (Du Pasquier, 2001 ) .
Fig. 1.6. Scheme of the defense strategies of arthropods to parasites and pathogens. (Rowley and Powell, 2007).
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Such alien microorganisms are recognized by a variety of pattern recognition molecules either free in the plasma or associated with various cell types. The cellular events consist of:
phagocytosis, nodule formation and encapsulation (Fig. 1.6). Phagocytosis is done by specialist
“professional” phagocytes while nodule formation removes large numbers of microorganisms from the hemocoel such that they become walled off by a sheath of cells. Encapsulation occurs when larger invaders or damaged self-tissues are recognized and become surrounded by a multilayer of hemocytes.
Bacteria and fungi are also killed by antimicrobial peptides (AMPs) or by intermediates of the prophenoloxidase cascade.
Changes of phenoloxidase activities depending on the developmental stage and age of the insects have been studied (Saltykova et al., 2003). Lectins and complement-like factors may act as recognition molecules and aid in the elimination of invading organisms (Rowley and Powell, 2007).
The immune system of Apis mellifera
The honey bees colony is a complex super-organism. A super-organism is composed of single individuals that together have a functional organization which is the formal definition of organism (Wilson e Sober, 1989 ). Each honeybee is similar to a single cell of the body of a superior organism:
The queen and the drone represent the gonads;
The ventilator bees and water bees are responsible of the thermoregulation (in the hive the temperature is between 35-37C° both in summer and in winter);
The foragers are responsible of the nutrition;
The nurses of the secretion;
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The hive sweepers and the necrophores of the excretion;
The skeleton function (honeycombs construction) is performed by the wax makers;
The cell multiplication is guaranteed by the brood;
The reproduction of the whole organism is left to the swarming;
The immune system is represented by the guardian bees that defend the hive from the external dangers. This function is also assigned to the bee-glue foragers because this resin has anti-microbial and anti-mycoses properties;
The hive communicates with the external world using the dance of honeybees.
A super-organism differs from an individual in that it is based on parallel operations and hence is less dependent on the precise functioning of all of its parts (Oster et al., 1978). The honeybees are in close contact and it is necessary a immune system of the colony. This immune system is represented by the social immune system.
Thus in the hive there are two types of immune system: the innate immune system (of the single individual) and the social immune system of the colony.
The innate immune system and the phenoloxidase
The bee is protected from most pathogens by its strong, waterproof cuticle. If the cuticle is
injured the pathogens meet the hemocytes (immune cells) in the hemolymph (the insect
equivalent of blood serum) that engulf any foreign invader at the wound site; then a chemical
cascade initiating with the enzyme phenoloxidase melanizes the clot into an inert and
impermeable barrier (Oliver, 2010). Hemocytes represent the cellular immunity (Wilson-Rich et
al., 2009), and perform a number of key actions, including the initiation of wound repair/blood
coagulation to prevent pathogen entrance into the main body cavity termed the hemocoel. If this
barrier is breached, the blood cells in the hemocoel can to perform phagocytosis and digest small
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invaders such as protozoans, bacteria, fungi, and viruses and incorporate multicellular parasites in a thick wall of hemocytes, in a process termed encapsulation (Rowley and Powell, 2007).
There are five types of hemocytes in the queen bees: pro-leukocyte, leukocyte, hemocytes type 1, 2 and 3 (Vecchi et al., 1977) (Fig. 1.7-1.8).
Fig. 1.7 Light microscopy picture of the hemocytes in the queen bee hemolymph: a. hemocytes type 1, l. leukocytes, p. pro-leukocytes (Vecchi et al., 1977, Tav I) Giemsa 960X
A. B.
Fig. 1.8 Light microscopy picture of the hemocytes in the queen bee hemolymph: A) hemocytes type 2. B) l.
leukocytes, c. hemocytes type 3. (Vecchi et al., 1977, Tav II) Giemsa 960X
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The hemocytes type 1 are present in the young queen bees with percentage 13-18% of cells but decrease with the age until 4%, they are big round cells, with a difficult to localize nucleus and a basophilic cytoplasm. The hemocytes type 2 and type 3 represent a higher percentage of the immune system cells, respectively 9.5-12.7% and 0.5-6%. The hemocytes type 2 are big cells with abundant basophilic cytoplasm and a central round nucleus. The hemocytes type 3 are ellipsoidal cells with a basophilic cytoplasm and a central oval nucleus. The pro-leukocytes are present in high percentage 45-50% in young queen (3-4 month) and are small cells with a big nucleus and an acidophilic nucleoplasm, and a little strip of basophilic cytoplasm. The leukocytes in the flutter queens are the most present hemocytes in the hemolymph but the morphology can vary among them. The leukocytes have a basophilic cytoplasm but in small quantity and the nucleus has the shape of the cell and is central.
In the worker honeybees (Szymaś et al., 2003), haemocytes were classified as plasmatocytes,
granular haemocytes, or other. Plasmatocytes were small round cells with a compact, round
nucleus and thin, hyaline neutrophilic or pale-basophilic cytoplasm or in which the cytoplasm
was not visible (Fig. 1.9). Granular haemocytes were large oval or ellipsoidal cells with a
granular nucleus and a vacuolated cytoplasm. Their cytoplasm was neutrophilic or pale-
basophilic and occupied most of the cell. Other types of haemocytes were large (from several
times to twelve or more times bigger than the plasmatocytes). They represented less than 10% of
the total number of haemocytes (Szymaś et al., 2003).
18 Fig.1.9 Haemocytes of young worker honey bees (magnification 1000 ×). G. granular haemocytes; P.
plasmatocytes; O. other haemocytes. From: SZYMAŚ et al., 2003.
The pro-hemocytes are the stem cells of the circulatory system and produce the other types of hemocytes. The granular hemocytes are the first cells that recognize a foreign body and have a role in the coagulation and the healing from the injuries. They release chemotactic factors in the hemolymph with the function to attract the plasmatocytes. The plasmatocytes are the hemocytes that perform the phagocytosis.
A lack of proteins causes a significant increase in the percentage of granular haemocytes, a significant decrease of other types and a lower metabolic activity (SZYMAŚ et al., 2003).
Phagocytosis is a simple form of defence and is associated with the opsonisating activity of
insect lectins that mark an invading parasite for the phagocytosing hemocytes (Scmid-Hempel,
2005). The phagocytosis comprises three events: recognition, engulfment, and destruction of
pathogens, apoptotic cells or infected cells. Lectins are considered simple recognition molecules
(Gillespie et al., 1997). The hemocytes release chemicals that penetrate the cell nucleus, and
cause them to up-regulate certain immune response genes, which then transcribe RNA
messengers that exit the nucleus, and then move to the ribosomes to translate the genetic
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instructions into antimicrobial peptides. This is called the “the induced” response. It appears that these peptides can be passed to nest mates to confer them resistance prior to being infected . The antimicrobial peptides are largely produced in the fat bodies. So there would be less of this sort of response in forager bees, which don’t maintain their fat bodies (Oliver, 2010). Schmid et al.
(2008) found that in all three adult phenotypes (queen, worker and drone) the number of hemocytes is dramatically reduced in the early adult life. Besides the hemocytes, in the hemolymph, there is the pro-phenoloxidase (Lourenço et al., 2005). This enzyme needs a serine protease, alpha-chymotrypsin, in order to change into the active form (Laughton and Siva-Jhoty, 2011a). Phenoloxidase (PO) represents the humoral immunity (Wilson-Rich et al., 2009) and is a copper-containing enzyme which catalyses the hydroxylation of monophenol to o-diphenol and its oxidation to o-quinone, which can be bactericidal (Mason, 1955; Pye, 1974). When quinines are present in excess, the black pigment melanin is formed (Pye, 1974). In practice melanization is the process by which tyrosine is converted to melanin (Fig. 1.10).
Fig. 1.10 Conversion from tyrosine to melanin.
The phenoloxidase system plays an important role in insect immunity against some internal
metazoan parasites (Nappi, 1973). Phenoloxidase is an enzyme that exhibits a typical Michaelis-
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Menten kinetic. The kinetic parameters calculated for substrate oxidation are V
max= 0.62 Umg/protein and K
M= 0.17mM for L-Dopa, and V
max=0.55Umg/protein and K
M=0.095mM for dopamine. The catalytic efficiency, calculated by the relation V
max/ K
Mshows that the affinity of the enzyme for dopamine is about 1.6 fold higher than for L-Dopa (Zufelato et al., 2004).
Phenoloxidase functions only in the extracellular environment, but since it lacks a signal peptide to direct its secretion, phenoloxidase is released to the hemolymph only by cell lysis. Ascorbic acid can actively act as a regulator of reactions of the phenoloxidase system in insects (Saltykova et al., 2007). Phenoloxidase-mediated melanin synthesis leads to a local increase of free radicals and quinines, the mediators of proteins cross-linking and the precursors of the melanin polymer, playing a fundamental role in encapsulation of non-phagocytosed microbes and parasites (Nappi, 1972; Zufelato et al., 2004). Encapsulation is a method to kill microbes when hemocytes bind to an invader forming a multilayer capsule in order to exclude them from the circulation and kill them by asphyxiation. Melanin is eventually deposited onto the parasite and, when further hemocytes are recruited, this can lead to encapsulation, the formation of a melanised cell capsule around a foreign invader (Fig. 1.11).
Fig. 1.11 Heterotylenchus autumnalis (N) melanised and encapsulated in a larva of Orthellia caesarion 3 days after infection. C, hemocytic capsule; M, melanin; n, nuclei of hemocytes (Nappi, 1973a).
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Smaller items may be instead neutralized by the formation of nodules. (Schmid-Hempel, 2005).
Nodule appears to bind bacteria and yeasts via their surface molecules: lipopolysaccharides and
beta-1,3-glucans. Nodules are melanized, which occurs by a biochemical pathway that gives rise
to reactive oxygen species that cause damage to the microbes. In encapsulation or nodulation
responses melanisation made further damage to the invaders by free radicals (Lavine et al.,
2003). Furthermore the phenoloxidase contributes to the melanisation of the cuticle and its
sclerotization in the pupa (Zufelato et al., 2004, Laughton et al., 2011a). Hormonal treatment
during the fifth larval instar, the prepupae and the pupae, induces earlier activity of the
phenoloxidase, save the pyriproxyfen treatment which caused higher enzymatic levels in the
pupae (Bitondi et al., 1998). Laughton (2011b) found that larval bees have low levels of
phenoloxidase activity while the adult workers produce stronger immune responses than drones,
and a greater plasticity in immune investment. In the same paper Laughton found, furthermore,
that in adult workers immune challenge resulted in lower levels of PO activity but it could be due
to the rapid utilisation of the enzyme and the subsequent failure to replenish the constitutive
phenoloxidase. In workers, PO activity reached a plateau within the first week of adult life, in
queens enzyme levels continuously increased with age and in drones slightly declined with age
(Schmid et al., 2008). Schmid et al. (2008), hypothesize that the reduction of hemocytes in
foragers is not worker-specific but is a general phenomenon in all three honeybee adult
phenotypes. The adult honeybees do not abandon cellular immunocompetence but foraging bees
may play a similar role as vaccinated individuals in a population by providing a type of herd
immunity, and in their absence the disease resistance capacity of the group is likely
compromised (Wilson-Rich et al., 2008). The phenoloxidase activity changes in relation with
different diets supplied to the honeybees (Alaux et al., 2010) and in samples infected with
Paenibacillus larvae (Sagona et al., 2012).
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An important component of the innate immune system are antimicrobial peptides, which are a large class of small proteins that act on pathogens. Antimicrobial peptides circulate freely in the hemolymph, secreted by the major production source, the fat body (a liver-like organ), and in smaller amounts by hemocytes and some epithelial cells (Bulet et al., 2003). Some of them are ubiquitously present in the hemolymph, regardless of infection, while others are only detected in appreciable amounts upon microbial challenge. The antimicrobial peptide defensin is seen in honey bees in two forms. Both are suspected to disrupt the bacterial membrane by weakening the permeability barrier, causing partial membrane depolarization and consequent reduction in ATP production, killing bacteria within a minute for most species (Bulet et al., 2005). One particularly defensin is royalisin (Bilikova et al., 2001), named for its original discovery in bee royal jelly (Fujiwara et al., 1990), currently renamed defensin 1 (NP_001011616) (Klaudiny et al., 2005).
Defensin 2 (NP_001011638), distinctive because it is cysteine-rich, is generally different from defensin 1 except for a stretch of 47 residues over which they share 56% sequence identity (Altschul et al., 1990). Another antimicrobial peptide distributed in the honey bees is apidaecin.
It is a proline-rich peptide only 18 amino acids long and is found in several isoforms in bees: 1a, 1b, II, and possibly a fourth one called III (Li et al., 2006). It is thought to be a slow-acting peptide against mostly Gram-negative strains (Casteels et al., 1989) with a brood activity, but a correspondingly low specificity. Its mode of action is not entirely known yet, but the hypothesis is that apidaecins cross the outer membrane, then bind irreversibly to a component of the
periplasmic space or inner membrane (Li et al., 2006)
.The active version of apidaecin is
detectable in adults, while in the larvae it exists in an inactive, precursor form (Casteels-Josson et al., 1993). Another antimicrobial peptide of the honey bees is the abaecin, which is a longer proline-rich peptide (34 amino acids long) effective against a smaller number of Gram-positive and Gram-negative bacteria compared to the apidaecins. It appears to increase the
permeability of the outer membrane, thus allowing lysozyme to weaken the cell wall
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through its glycosidase activity (Casteels et al., 1990).
Another antimicrobial peptide is hymenoptaecin: 93 amino acids in length, with a unique 2- pyrrolidone-5-carboxylic acid at the amino-terminus. It has bactericidal activity against some bacteria of both Gram stains. By testing on E. coli, it was concluded that the mode of action was sequential permeabilization of the outer and inner membrane (Casteels et al., 1993). Apisimin is a new antimicrobial peptide (Bilikova et al., 2002) recently descovered.
Randolt et al. (2008) presented evidence that in vitro reared honey bee larvae respond with humoral reaction to aseptic and septic injury as documented by the transient synthesis of three antimicrobial peptides (AMPs) hymeniptaecin, defensin1 and abaecin. Randolt et al. (2008), said also that young adult worker bees react with the activation of prophenoloxidase and humoral immune responses (Randolt et al., 2008). Phenoloxidase, peptidoglycan recognition protein-S2, carboxylesterase and an Apis-specific protein (HP30) are induced specifically in adult bees (Randolt et al., 2008).
The lysozyme is a protein, an enzyme and occasionally is categorized as an antimicrobial peptide. The lysozyme is a normal constituent present costitutively in insect hemolymph and hydrolyzes the 1,4 glycosidic linkage of alternating units of N-acetylglucosamine and N- acetylmuramic acid residues that make up the peptidoglycan of the bacterial cell wall. In the honey bees three lysozyme genes are predicted (Evans et al., 2006). They are mainly active against Gram-positive bacteria because their cell wallis composed largely of peptidoglycan.
The insects express a set of complement-like proteins of which the best studied belong to a group
called thioester-containing proteins (TEPs). TEPs attach to target surfaces of pathogens by
forming covalent bond. Two other groups, Janus kinase (JAK)/signal Transducers and Activators
of Transcription (STAT) pathway, contribute to the expression of these proteins. Honeybees
have three TEP homologs and homologs of JAK/STAT pathway except for the initiating ligand
but it exists other proteins for this function.
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Although the Invertebrates don’t have immunological memory, Sadd et al. (2006) demonstrated that the invertebrate immune system is capable of responding in a specific manner to a previously encountered pathogen type. After a period of three weeks they demonstrated that the immune system is also functionally protective upon secondary exposure (Sadd et al., 2006).
The social immune system and the glucose oxidase
The ‘social immune systems’ result from the cooperation of the individual group members to combat the increased risk of disease transmission that arises from sociality and group living (Cremer et al., 2007).
The first step for the honeybees to defend the colony from parasites are hygienic behaviours.
Hygienic behaviour is defined as the ability to detect and remove diseased brood from the nest
(Wilson-Rich et al., 2009). For the American foulbrood, the bees have the ability to detect and
remove diseased brood before the causative organism, Paenibacillus larvae, reaches the
infectious spore stage in the diseased larvae (Wilson-Rich et al., 2009). Regarding to Varroa
destructor, bees are able to detect and remove pupae that are parasitized with this mite,
suspending the mite reproductive cycle (Spivak et al., 1998; Boecking et al., 1999; Spivak et al.,
2001). Another mechanism to defend themselves from Varroa is the grooming which is a
behaviour in which an insect cleans another one with the legs to eliminate parasites. Another
hygienic behaviour is the use of propolis by honeybees. Propolis is a tree resin that honeybees
use to disinfect the hive and to close open spaces. Regarding to chalkbrood the honeybees
exhibit significantly increased sensitivity to the odor of this disease based on
electrophysiological recordings of nerve impulses from the antennae. So the bees tend to uncap
dead brood and to remove dead brood from the hive (Wilson-Rich et al., 2009) (Fig. 1.12).
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Fig 1.12 Graphical representation of honey bee hygienic behavior. (Wilson-Rich et al., 2009)
Le Conte identified a set of genes involved in the social immune by analysing the brain trascriptome of highly Varroa-hygienic bees, who efficiently detect and remove brood infected with the Varroa destructor mite (Le Conte et al., 2011). Rueppel et al. (2010) found that honey bee foragers treated with prolonged CO
2narcosis or feeded with the cytostatic drug hydroxyurea showed an increased mortality but the surviving foragers abandoned their social function and removed themselves from their colony, resulting in altruistic suicide. A simple model suggests that altruistic self-removal by sick social insect workers to prevent disease transmission is expected under most biologically plausible conditions (Rueppel et al., 2010). Thermoregulation in general is used by the honey bees as a defence against diseases (Wilson-Rich et al., 2009). The
“social fever” in which many individuals increase their body temperature to kill the bacteria in the hive with the heat. Simone et al. (2009) analyzed the use of resins of the honey bees. The
Detection and removal of mite-infested brood, usually after mite has started laying eggs
Detection and removal of diseased brood before disease forms infectious spores
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individual bees in resin-enriched colonies in the field are able to invest less energy on immune function for two divergent immune-related genes, and this effect is conceivably due to decreased bacterial loads (Simone et al., 2009).
The second step for the honeybees to defend the colony from parasites is the capacity to sterilize all surface with antimicrobial secretions in their saliva. In the royal jelly, in particular, there are (Scarselli et al., 2005):
royalisin, an antimicrobial peptide against Gram-positive bacteria and fungi (Fujiwara et al., 1990);
the jelleines, are an antimicrobial family of peptides against Gram-positive, Gram-negative bacteria and yeasts (Fontana et al., 2004).
Furthermore there is an enzyme secreted by the hypopharyngeal gland: the glucoso oxidase (Takenaka et al., 1990, Ohashi et al., 1999).
Fig. 1.13 Glucose oxidase with its two subunits depicted in dark and light blue, while the FAD coenzyme is depicted in pink. Image by Goodsell (2006), the Scripps Research Institute.
27
The glucose oxidase (Fig. 1.13) is a chromoprotein enzyme, a flavoprotein (Coulthard et al., 1945) with a molecular weight of about 149KDa and with the prosthetic group that has been identified as alloxazine-adenine-dinucleotide (Keilin et al., 1952a); there are two prosthetic groups in each protein. The properties of the glucose oxidase are listed in table 1.2.
Table 1.2. Properties of glucose oxidase from A. Niger (table from Wong et al., 2008)
Properties Values
Molecular weight
150–186 kDa (Swoboda and Massey, 1965; Nakamura and Fujiki, 1968; Tsuge et al., 1975)
UV–VIS absorption
280: 380: 450 (nm) (Tsuge et al., 1975) 11.5: 1: 1.03 (ratio) (Tsuge et al., 1975)
Extinction coefficient
270,000 M−1 cm−1 (at 280 nm)
25,180–28,200 M−1 cm−1 (at 450 nm. Swoboda and Massey, 1965; Johnson et al., 1989)
21,600 M−1 cm−1 (at 452 nm. Nakamura and Fujiki, 1968) Specific activity (pH 5.6,
25–37°C)
80–172 μmol glucose/min/mg enzyme (Swoboda and Massey, 1965; Tsuge et al., 1975)
Km (Michaelis constant, with respect to glucose)
198–248 mM (pH 5–7, 20–30°C, oxygen; Bao et al., 2001) 110–120 mM (pH 5.6, 0–38°C, oxygen; Gibson et al., 1964) 50–74 mM (pH 5.5, 15–30°C, oxygen; Nakamura and Ogura, 1968b)
33 mM (pH 5.6, 25°C, oxygen; Swoboda and Massey, 1965)
41.8 mM (pH 6.86, 25°C, benzoquinone)
28
Properties Values
Temperature range 20–50°C (Gouda et al., 2003)
pH range
4–7 (Keilin and Hartree, 1947; Nakamura and Fujiki, 1968;
Bao et al., 2001)
Inhibitors
Ag+, Hg2+ and Cu2+ ions (µmol) (Nakamura and Ogura, 1968a; Toren and Burger, 1968)
Arsenite, p-chloromercuribenzoate, phenymercuric acetate (mmol) Nakamura and Ogura 1968a) and others
Isoelectric point (pI) 4.2 (Pazur and Kleppe, 1964)
The glucose oxidase is produced by the hypopharingeal glands and its activity is highest in nurse bees (Fig. 1.15). It also present in the honey (White jr, 1966), in the royal jelly (Takenaka et al., 1990; Furusawa et al., 2008), so it could be considered ubiquitous in the hive. The glucoso oxidase as described in honeybees by Schepartz and Subers (1964) is involved in the reaction where the β-glucose (Schepartz, 1965a) with oxygen and water, is converted in hydrogen peroxide and gluconic acid (Fig. 1.14) (Keilin et al., 1948; Keilin et al., 1952, Schepartz, 1965b).
The initial product of glucose oxidation is D-gluconolactone and it is a weak competitive
inhibitor of the glucose oxidase (Wilson and Turner, 1992). The D-gluconolactone hydrolyses
spontaneously to gluconic acid (Takahashi and Mitsumoto, 1963). The rates of oxidation of the
two isomers of glucose are β:α=100:0.64 so the study of the catalytic activities of glucose
oxidase requires careful correction for mutarotation of α-glucose (Keilin et al., 1952a).
29 Fig. 1.14 The reaction in which the glucose oxidase is involved. GOD= glucose oxidase, FAD= flavin adenine dinucleotide, FADH2= flavin adenine dinucleotide reduced
Fig. 1.15 Changes of glucose oxidase activity in hypopharingeal gland and gluconic acid content. (Takenaka et al., 2133).
30
Either these products contribute to the disinfection of the colony and the avoidance of the contamination of pathogens and diffusion of diseases (Alaux et al., 2010). Natural full-density honey contains no hydrogen peroxide (<0.3ppm), but measuring the increase in acidity during long term storage, the system produces in full-density honey about 0.002-0.012µg H
2O
2/g honey/
hr (White Jr et al., 1964). White et al., (1963), found that the inhibine, the antibacterial substace studied before 1963 and reported in honey, to be hydrogen peroxide produced in the inhibine assay by the natural glucose oxidase in the honey. More than about 30 micrograms of peroxide per plate (about 0.0002%) prevent bacterial growth (White Jr et al., 1966). Gluconic acid is the most aboundant acid of honey (Stinson et al., 1960). Pulcini found different total gluconic acid (TGA) content in Italian unifloral honeys (Pulcini et al., 2004).
The glucose oxidase activity changes in relation with different diets supplied to the honeybees (Alaux et al., 2010) and in samples infected with Paenibacillus larvae (Sagona et al., 2012).
Honeybees gut microbiota
Symbiosis is the interaction between two or more different biological species that work synergistically to maintain proper nutrition, health and immunity. In this perspective, the gut microbiota considered as a different organism, could contribute to maintainance of the immunity of the honeybees. The pH of the gut of honeybees is highly acidic but can change depending on the ingested food. The microbial community of the social stomach has a role in the preservation of food completing the functions of the salivary system.
The bacterial group (table 1.3) is the same, independently of geographical location, for all the
honeybees. This suggests that this group is coevolved with honeybees (Anderson et al., 2011).
31 Table 1.3 Non pathogenic microorganisms commonly sampled from the honey bee alimentary tract and hive environment. A. Considered the “core” gut microbiota. (Anderson et al., 2011).
The adult gut includes Lactobacillus (Lactobacillaceae), Bifidobacterium (Bifidobacteriaceae),
Acetobacteriaceae, Gammaproteobacteria group (Enterobacteriaceae and Pasteurellaceae),
Simonsiella (Neisseriaceae) and Bartonella (Bartonellaceae). Lactobacillus (Firmicutes) which
accounted for over 70% of the 16S RNA gene sequences and Bifidobacterium are estimated to be
2.8-8.4% of total bacteria in honey bees. These bacteria are involved in immunomodulation,
interference with enteric pathogens and the maintenance of a healthy microbiota. Within
Lactobacillus spp. Lactobacillus johnsonii has been identified (Audasio et al., 2011). One of the
group of Acetobacteriaceae in the honey bees is the genus Gluconobacter (Ruiz-Argueso et al.,
1973; Lambert et al., 1981). The Gluconobacter is an obligately aerobic bacteria adapted to
highly acid environments rich in sugar. It use Krebs cycle enzymes for the incomplete oxidation
of sugars, ethanol, and polyalcohols, often producing large amounts of gluconic acid as the final
product (Anderson et al., 2011). Gluconobacter is equally distributed in the alimentary tract, that
32
is the main reservoir, and the rest of the body. Lactobacillus spp. are mainly located in the mesointestine, and a significant number is on the body surface (Ruiz-Argueso et al., 1975). The members of the Gammaproteobacteria group are facultative anaerobes and ferment sugars to produce lactic acid and various other end products that may reduce nitrates to nitrites suggesting a potential function in nitrogen methabolism within the gut. The genus Simonsiella is a strict aerobe that generates filaments of eight or more cells that adhere tightly to host epithelial cells.
The genus Bartonella is a facultative intracellular parasite and has the ability to derive carbon and energy from the catabolism of amino acids rather than glucose.
Larval bees don’t partake in social stomach and thus have fewer niches for microbial establishment. Bacillus spp. can sporulate and produce bacteriocins or bacteriocin-like compounds directed towards species which may want to occupy the same niche.
Evans et al. (2006) suggested a tradeoff in social insect colonies between the maintenance of potentially beneficial bacterial symbionts and deterrence at the individual and colony level of pathogenic species.
Honeybees proteases
The proteases are involved in various physiological processes, such as digestion, development and defense responses. The proteases are often synthesized as zymogens, which require proteolysis at a specific site for activation. Serine proteinases play a role in many diseases and in the proteolytic cascades involved also in invertebrate immunity (Cerenius et al., 2010). To avoid undesired proteolysis that can lead to numerous pathological processes, the organism possesses many inhibitors that function by specifically inactivate these enzymes (Bania et al., 1999).
Polanowski et al., 1992 investigated the inhibition of trypsin, chymotrypsin, neutrophil elastase and cathepsin G, and pancreatic elastase from the hemolymph of 14 insect species in six orders.
These analyses showed great diversity in terms of both total proteinase inhibitory capacity and
33
specificity. Furthermore the highest total inhibitory capacity was found in the larval hemolymph of species in the beetle family Tenebrionidae and the lowest in that of an adult coreid bug, Acanthocephala femorata (Polanowski et al., 1992). Zou et al. (2006) identified 44 serine protease (SP) and 13 serine protease homolog (SPH) genes in the genome of Apis mellifera.
Grzywnowicz et al. (2009) described how protease and protease inhibitor activity patterns vary in A. mellifera during ontogenesis, with season, and in relation to caste and sex. Bania et al.
(1999) demonstrated that the larval hemolymph of the honey bee contains polypeptides that are
able to inhibit the catalytic function of several serine type proteases (anti bovine chymotrypsin
and human cathepsin G). The chymotrypsin inhibitor-1 (AMCI-1) was characterized by H NMR
spectroscopy (Otlewski et al., 2001). Worker, queen and drone eggs and the body surface of the
queen have a very low proteolytic activity in contrast to the body surface of the drone and the
worker that have a high proteolytic activity in spring and in summer, respectively (Strachecka et
al. 2008). In the queens all the catalytic protease types are present: asparagineand cysteine
proteases at pH=2.4; cysteine proteases and metalloproteases at pH=7; and serine protease at
pH=11.2 (Strachecka et al., 2011). The larvae and pupae have higher proteolytic activity than the
adult, and the dead honey bees have higher proteolytic activity than the living honey bees
(Strachecka et al. 2008). The adaptation of herbivore insects, such as honey bees, to plant
protease inhibitors was studied by Jongsma and Bolter (1997). Ramesh et al. (2005) concluded
that nurse bees fed with a pollen diet containing at least 1% of Soybean trypsin inhibitor are poor
producers of larval food, potentially threatening colony growth and maintenance.
34
Honey bee diseases and pests
All the organisms are subject to infestation or attack by their predators and Apis mellifera is no exception. Many vertebrates are natural enemies of honey bees. These include amphibians, reptiles, birds and mammals. Native bees are generally adapted to the normally present pathogens but the unnecessary importation of foreign bee species and races have disastrous effects in spreading bee diseases and parasites. The enemies of the honey bees include microbial diseases, parasitic bee mites, insects and vertebrates.
Bacterial diseases
The main bacterial diseases are American foulbrood, European foulbrood and Parapest.
American foulbrood (AFB)
The American foulbrood is a brood pathology caused by Paenibacillus larvae (Fig. 1.16)(K.
Antύnez et al., 2007). AFB is classified on list B of the Office International des Epizooties (OIE), the world Organization for Animal Health. List B diseases are defined as transmissible diseases which are considered to be of socio-economic and/or public health importance within countries and which are significant in the international trade of animals and animal products (de Graaf et al., 2006). P. larvae is a spore-forming bacterium, Gram+, mobile, facultative anaerobe.
It measures 2-5 µm of length and 0.5-0.8 µm of width (Giordani et al., 1982). The spores, 0.6 x 1.3 µm, are oval brilliant corpuscles, and represent the reproductive and resistance form.
Fig 1.16 Paenibacillus larvae. From ASM MicrobeLibrary.org (photo by Donald Stahly).
35
P. larvae is introduced in the larvae as spores with the food and infects the larvae of A.mellifera in the first 24 hours after hatching. When the hive cells are sealed the spores germinate in semi- anaerobic condition and the vegetative form of the bacteria kills the larvae (Rauch et al., 2009).
In Paenibacillus-infected larvae, all midgut epithelial cells die, but Paenibacillus infection does not cause increased programmed cell death (Gregorc and Bowen, 2000). Gregorc and Bowen (1998) observed cell necrosis in the midgut epithelium accompanied by increasing cell vacuolization and nuclear pyknosis following per os inoculation of P. larvae. Degradation of hemocytes, salivary glands and other tissues was also observed from Gregorc and Bowen (1998).
Dancer et al. (1997), studied the gross protease activity of pathological samples of American foulbrood-infected cadavers from several UK sources. They found that in all cases the bulk of the activity was caused by neutral proteases (optimum pH ca. 6.8, optimum temperature 60- 65°C) that were inhibited by chelating agents such as EDTA and 1,10 phenanthroline (indicating that the were probably metalloproteases). Antúnez et al. (2009) identified two different proteases patterns (PP1 and PP2) in a collection of P. larvae isolates from different geographical locations.
Antúnez et al. (2011a) demonstrated the presence of a metalloprotease inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted in the external growth medium.
This protein was produced in vivo during infection of honeybee larvae and it was able to
hydrolyze milk proteins as described for P. larvae, suggesting it could be involved in larval
degradation (Antúnez et al., 2011a). Ten different proteins (involved in transcription, translation,
metabolism, cell envelope, transport, protein folding, degradation of polysaccharides and
motility) in the P. larvae secrete were identified which resulted highly toxic and immunogenic
when larvae were exposed using an experimental model (Antúnez et al., 2010). Proteolytic
activity was detected also in pollen contamined with P. larvae by the milk coagulation test
(Hrabák and Martínek, 2007). Antúnez et al. (2011) identified an enolase as a potential virulence
36
factor secreted by P. larvae. Studying five day old healthy and P. larvae-infected bee larvae, Chan et al. (2009) observed an increased level of the immune factors prophenoloxidase (proPO), lysozyme and hymenoptacein in samples infected. The gene Abaecin shows significant up- regulation 24 hours after oral inoculation with P. larvae, precisely when the bacterium surmounts the mid gut epithelia of bees (Evans, 2004).
Died larvae become creamy or dark brown, glue-like larval remains and perforated cells represent the clinical symptoms of American foulbrood (de Graaf et al., 2006) (Fig. 1.17). Very characteristic symptoms of AFB are also the decomposition of the pupal stage in which the tongue protrudes from the head and a peculiar bad smell. Lindström (2008a) developed a mathematical formula for estimating the minimum number of bees in a sample to detect clinical disease. In table 1.4 different techniques to identify Paenibacillus larvae are shown.
a) b)
Fig. 1.17 Characteristic symptoms of AFB: a) died larvae become creamy or dark brown, glue-like larval remains, b) perforated cells.
37 Table 1.4 Different techniques to identify Paenibacillus larvae. From: de Graaf et al., 2006.
Technique Principle Samples Advantages Disadvantages
Cultivation Germination and growth of Paenibacillus larvae spores on solid medium
Brood, honey, adult bees, pollen, wax, hive debris
Detection of P. larvae in bee products facilitates tracing infection sources Very suitable for American foulbrood (AFB) detection programmes Permits quantification of the spore-load
Allows to test spore viability
Requires an additional identification step
of suspect P. larvae colonies Semi-selective media usually required to avoid contamination with other bacteria
Biochemical profiling
Identification of the species of bacteria based on the carbohydrate acidification profile, the catalase test and the casein hydrolysis plate test
Bacterial colonies
Is a traditional microbiological approach
that can be performed in most microbiology laboratories
Requires a first step of isolation and cultivation of bacteria
Results for a full profile are available after 2 to 3 weeks, although identification
as P. larvae can be based on Gram reaction, catalase reaction and colony morphology
Phage sensitivity test
Plaque formation in a semi-solid medium as a result of bacterial cell lysis
Bacterial colonies
Easy and simple test to perform
Rapid diagnosis of AFB Low cost
Requires a first step of isolation and cultivation of bacteria
PCR Amplification of
specific bacterial DNA using a single primer set
Bacterial colonies, brood, honey
Permits rapid confirmation without
cultivation step starting from diseased brood
Needs sophisticated equipment
Nested PCR Amplification of specific bacterial DNA by using external and internal primer
sets in subsequent reactions
Brood, honey, adult bees
Permits rapid confirmation starting from
a broad range of samples Because of its high sensitivity very suitable
for AFB detection programmes
Needs sophisticated equipment Can identify levels of P. larvae that are below those likely to be important for disease
Can identify the presence of dead spores or spores that fail to germinate,
both not important for disease Nested PCRs are more prone to contamination
Microscopy
Morphological identification of P. larvae spores
Brood Smears can be made in the field
and forwarded to the laboratory for examination
Rapid diagnosis of AFB Low cost
Only for confirmation of clinically diseased larvae
Immunotechniques Different tools for identification based on specific antigen–
antibody interactions
Brood The commercialized lateral flow device permits easy confirmation of clinical AFB in the field
Rapid diagnosis of AFB
Utility of each test is highly dependent
on the specificity of the antibody preparation used
Rauch et al. (2009) concluded that the time of larval death and the virulence on the larval level
are negatively correlated to the time to colony death and the virulence on the colony level. The
38
transmission of AFB (American foulbrood) between apiaries occurs within 1 km from clinically diseased colonies, but is significantly lower at 2 km or more when colonies dead from AFB are allowed to be robbed out (Lindström et al., Apidologie 2008). Vertical transmission of Paenibacillus larvae may occur during reproductive swarming (Fries et al., 2006). Gillard et al.
(2008) studied bee samples collected from the brood nest, honey chamber, and edge frame where they detected all colonies showing AFB clinical symptoms and one quarter of samples collected from colonies without AFB clinical symptoms were positive for P. larvae. Contaminated honey can act as an environmental reservoir of this microorganism and this suggest that less spores may be needed in honey to produce clinically diseased colonies (Lindström et al., 2008b).
A kit (VITA) can be used to identified the bacteria of AFB (Fig.1.18).
a. b.
Fig. 1.18 Commercial kit for beekeepers to make the diagnosis of AFB. a. The kit b. in this image T is the same of C indicating the presence of P.Larvae spores are present.
Spivak and Reuter (2001a) showed that honey bee colonies selected for hygienic behaviour demonstrated resistance to American foulbrood disease. The scientists and the beekeepers have tried many methods to eliminate this dangerous disease. Antibiotic are bacteriostatic (H.
Katznelson, 1950), radiations inactive the bacteria (Zenaida M.DeGuzman et al., 2011), plant’s extract, propolis and essential oils have effect on P. larvae (K. Antύnez et al., 2008, M.J.
González et al., 2010 Jaroslav Flesar et al., 2010, N. Roussenova, 2011). In the last years some
39
bacteria were tested against P. larvae; in particular those microorganisms that seem to have an effect are: selected strains of aerobic spore-forming bacteria isolated from apiarian sources (Alippi and Reynaldi, 2006), Bacillus subtilis isolated from honeybee gut and honey samples (Sabaté et al., 2009), novel lactic acid bacteria originating from the honey stomach (Forsgren et al., 2010) and lactic acid bacteria isolated from fermented materials (Yoshyama et al., 2013).
European foulbrood (EFB)
European foulbrood is a brood pathology generally considered less virulent than AFB. The pathogenic bacterium of EFB is Mellissococcus plutonius. The bacterium is Gram-positive and does not form spores (Fig. 1.19). It is lanceolate in shape and occurs singly, in chains of varying lengths, or in clusters; it measures: 0.5-0.7pm in width by 1.0 pm in length (Ritter and
Akratanakul, 2006). Melissococcus plutonius has a permanent form, does not form spores but capsules which are less resistant than the spores of P. larvae.
Fig. 1.19 Scanning electron micrograph of Melissococcus plutonius. The bar represents 1 µm. Image from Forsgren, 2010 (photo by Ingemar Fries).
The bacterium generally attacks only younger larvae in uncapped cells (Reybroeck et al., 2012).
The diseased larvae die when they are four to five days old, or in the coiled stage. The infected
larva moves in the brood cell, and instead of the normal, coiled position, the larva dies displaced
in its cell (Forsgren Eva, 2010). The color of the larvae changes from pearly white to brown and
finally, grayish black (table 1.5). Larvae killed by EFB, in contrast to AFB scales, do not adhere
40
to the cell walls and can be removed with ease. The odour can vary depending on the kind of other bacteria involved (Bacillus alvei, Streptococcus faecalis, Achromobacter eurydice).
Oxytetracycline has been demonstrated effective against EFB (Reybroeck et al., 2012).
Table 1.5 Comparative symptoms of AFB and EFB. From Alippi, (1999).
Parapest
The para-foulbrood is a brood disease which have symptoms halfway between the European and
American foulbrood. The pathogenic bacterium of para-foulbrood is Bacillus para-alvei. This
bacterium attacks the larvae and kills them when the cells are capped. The operculum of cells
with died-larvae appears dark, sunken and oily. The died-larva appears dark red and has a
characteristic smell.
41
Virus diseases
Viruses are microorganisms which don’t have a cellular structure and depend on the host-cell.
With the arrive of the Varroa destructor in Italian apiaries, viruses found a new carrier and a new host where to reproduce themselves before entering in the honeybees.
Some of the viruses found in apiaries are (Contessi, 2007):
Black queen cell virus- It is coupled with Nosema. This virus attacks the queen pupae which die and become black.
Virus Y and Filamentous virus- They are coupled with Nosema.
Bee Virus X (BVX)- It is coupled with the agent that causes amoebiasis.
Cloudy wing viruses (CWV)- It is transmitted by direct contact or by air. In some cases it is associated to the Varroa mite.
Chronic bee-paralysis virus (CPV)- In some cases bees which can’t fly, with shaking walk and that die in front of the hive, can be observed. Symptoms are the inflated abdomen and the wings arranged to “K”. This kind of disease was called “Pain of the forest”. In some cases bees become black and lose the wools. After some days bees with shaking walk that die can be observed. This kind of disease was called “Black pain”.
Apis iridescent virus (AIV)- It causes the “glomere” disease.
Slow paralysis virus (SPV)- It is associated with Varroa.
Acute paralysis virus (APV)- It attacks the fat bodies of the honey bees but if it arrives in the hemolymph it kills the bees. It is transmitted by the Varroa.
Deformed wing virus (DWV)- It is transmitted by the Varroa.
Arkansas, Berkeley, Kashmir and Picorna viruses were discovered in Italian apiaries (Cersini et al., 2013) but are still little known.
Sac brood virus (SBV)- It is a brood disease.
42
Deformed wing virus (DWV)
Deformed wing virus (DWV) of Apis mellifera is a virus closely associated with characteristic wing deformities, abdominal bloating, paralysis and rapid mortality of emerging adult bees (Fig.
1.20) (Lanzi et al., 2006).
Fig. 1.20 On the left honeybee infected with DWV, on the center Varroa destructor mite, on the right an healthy honeybee.
DWV produces a 30-nm icosahedral particle consisting of a single positive-stranded RNA genome and three major structural proteins. DWV and SBV are similar to mammalian picornaviruses. This virus is transmitted by the ectoparasitic mite Varroa jacobsoni (Bowen- Walker et al., 1999). Shen et al. (2005) demonstrated that in mite-infested bee pupae DWV had amplified to extremely high titres with viral genomic RNA, from activated viral replication.
Francis et al. (2013) observed as the DWV titres increased, the genetic diversity of DWV
decreased ultimately leading to a single high-virulent species. Quantification of viral genome
equivalents revealed that mites capable of inducing an overt DWV infection contained 10
10-10
12genome equivalents per mite (Gisder et al., 2009). Yang et al. (2005) suggested that
parasitisation by Varroa suppresses the immunity of honey bees, leading to activation of a
43
persistent, latent viral infection. Colony size, the number of workers with wing deformities and V. destructor infestation levels constitute predictive markers for winter colony losses in this order of importance and ease of evaluation (Dainat and Neumann, 2013). Investigations on the infection of DWV suggest a connection between diet (lowest in pollen fed bees), protein levels and immune response and indicate that colony losses might be reduced by alleviating protein stress through supplemental feeding (DeGrandi-Hoffman et al., 2010).
Sac brood virus (SBV)
Sac brood disease is caused by Morator aetotulas virus and is a relatively common disease.
Several reports indicate that the nurse bees are the vectors of the disease because larvae are infected via brood-food gland secretions (Ritter and Akratanakul, 2006). It has an hexagonal shape of 28 nm and contains RNA. This virus is located in the cytoplasm of cells in larval fat and muscle tissue and in the cytoplasm of the fat body cells of adult bees infected with virus (Morse, 1978). The infectivity is sensitive to heat. The characteristic saclike appearance (Fig.1.21) is the result of a disruption of the normal molting process and the darkened head derives from hardening and differentiation of the larval skin (Morse, 1978). 10
7-10
8particles of sac brood virus are necessary to produce apparent sac brood virus disease in a two-day-old larva. When removing the infected larvae from the cells their skin appears quite tough and its content is watery, and the larva appears as a small, watery sac (Ritter and Akratanakul, 2006).
Fig. 1.21 Sac brood virus. The larva appears as a small watery sac. From Williams (2000).
