BACTERIAL CONTAMINATION OF HAEMOLYMPH IN EMERGING WORKER HONEYBEE (APIS MELLIFERA L) PARASITIZED BY VARROA DESTRUCTOR

Varroa destructor is an obligatory ectoparasite of the honeybee (Apis mellifera L). The mites use their piercing month parts to suck out hemolymph from immature and adult bees caused direct damage (morphological, physiological abnormalities) and indirect damage due to microbial pathogens. The aim of this work was to research the bacterial microflora in hemolymph of emerging healthy and parasitized worker honeybee by Varroa destructor. For the isolation and identification of the bacteria, the morphological and biochemical tests were done. The results showed that the haemolymph of the healthy worker (Apis mellifera L) is free of bacteria. When the V.destructor breaks the cuticle, the microorganisms invade the heamolymph. The infested worker honeybees harbored a total 9 strains belonging to 6 strains of Bacillacea (66,66%), 1 of Peanibacillacea (11,11%) and 2 of Enterobacteriacea (22,22%). Based on the Gallery API 20 E (Bio-Merieux), the genera of Bacillacea and Peanibacillacea included Bacillus licheniformis (4 strain), Bacillus mycoide (1strain), Bacillus coagulans (1 strain) and Brevibacillus chohinensis (1 strain) respectively. Bacillus licheniformis was for probably the most frequent species. The Enterobacteriaceae included Aeromonas hydrophila and Pantoa sp.


INTRODUCTION
Varroa destructor (Anderson and Trueman, 2000) an obligatory ectoparasite of the honeybee (Apis mellifera L), has caused severe damage to populations of this species world-wide in recent years (Le Conte et al., 2010). The direct negative effect of the Varroa on honeybee has been well documented (Weinberg and Madel, 1985;Daly et al., 1988, Wienands andMadel, 1988;Marcangeli et al., 1992;Bowen-Walker and Gunn, 2001;Contzen et al., 2004;Yang and Cox-Foster, 2005;Belaïd and Doumandji, 2010;Belaïd et al., 2017). However, in the recent years, scientists have diverted their attention towards the indirect effect by virus transmission, the foulbrood diseases and fungal infection (Hrabak, 2003;Benoit et al., 2004;Tentcheva et al., 2004, Hamdi et al., 2011. Tentcheva et al., (2004) reported that the infection of bees with the Deformed Wing Virus (DWV) was strongly linked to the presence of Varroa. De Rycke et al., (2002) reported that Varroa destructor was capable of transporting spores of Paenibacillus larvae (the American foulbrood agent) to the surface of its body, thus allowing the parasite to participate in its propagation. The Fungi or spores of fungi are found on the surface of V. destructor (Aspergillus flavus, Penicillium multicolor, Penicillium simplicissimum, Mucor ramosissimus, Mucor indicu, Mucor hiemalis and Ascosphaera apis (Benoit et al., 2004). The cuticle itself constitutes an excellent barrier against parasite invasion. However, the damaging host integument during the feeding behavior of Varroa rendered the bees vulnerable to the microbial infections (Kanbar and Engels, 2005). To our knowledge, there are no reports about the bacterial microflora in heamolymph of honeybee parasitized by Varroa destructor. In this paper, the bacteria enable to be transmitted into honeybee haemocoel was investigated.

MATERIAL AND METHODS
Healthy and parasitized preemerging honeybees (Apis mellifera L) were collected from the brood of the apiary of Tizi Ouzou in early summer 2015. 1 µl of haemolymph of the samples were diluted in 9µl sterile normal saline (1:10 µl) immediately vortexed, then the haemolymph solution was plating on nutrient agar plates with the help of sterilized loop and incubated at 37° C. The plates were prepared in duplicate. Each different colony was subcultured to obtain pure culture. Selected strains were initially characterized by cell morphology and Gram's, endospore staining using the standard procedures. Primary identification was carried out according to Bergey's Manual of systematic Bacteriology (Holt et al., 1994). Biochemical characteristics were tested with API 20E galleries (Biomerieux).

RESULTS AND DISCUSSION
The preliminary results about the occurrence of bacteria microflora in haemolymph of emerging healthy and parasitized worker honeybee (Apis mellifera intermissa) by Varroa destructor were shown in table 1, table 2 and Fig  1 ( A, B).

Table1.
Biochemical characteristics of isolated bacteria from the haemolymph in emerging worker honeybee parasitized by V. destructor.   In our work, the haemolymph of the healthy emerging worker honeybee was free of bacteria. According to Tubiash et al., (1975), it was generally assumed that the circulatory system of healthy animals was sterile. The body surface of adult honey bees is relatively free of bacteria, likely due to grooming behavior (Gilliam, 1997). According to Keller et al., (2013), the pre-adul honeybee was almost sterile systems. In invertebrates, the circulating haemocyte has a major role in the protection of the animal against aggressive microorganisms by participing in recognition, melanisation, phagocytosis and cytotoxicic activities (Jiravanichpaisal et al., 2006). The presence of bacteria is usually considered to be a sign of disease (Tubiash et al., 1975). Varroa bee hive attack is a serious and common problem in beekeeping. The mite gets attached to the body of the bee and brood and weakens the bee by repeated sucking of haemolymph (Vanikova et al., 2015). When the Varroa destructor breaks the cuticle, the microorganisms invade the heamolymph (Kanbar and Engels, 2005).
To our knowledge, this work was the first report studying the role of the mite as vector to bacterial microflora in the haemolymph of honeybees.
In the study, the results showed that the Bacillacea was the most frequently present in heamolymph of worker honeybees (Apis mellifera L) followed by Enterobacteriaceae and Peanibacillacea. According to De Rycke et al., (2002), Varroa destructor could play a role in the transmission of Paenibacillus larvae spores, Bacillus (formerly known as Bacillus larvae) responsible for American foulbrood from infected to healthy bee colonies. Hrabak (2003) identified the genus Staphylococcus albus and Enterobacter cloacae associated to the ectoparasite mite. Tsagou et al., (2004) isolated bacterial strains from Varroa destructor belonging to Bacillacea (Bacillus sp) and Micrococcaceae. Hubert et al., (2015) found Morganella sp, Enterococcus sp, Pseudomonas sp, Rahnella sp, Erwinia sp and Arsenophonus sp. Maddaloni and Pascual (2015) reported the occurrence of Bacillus subtilis, Pseudomonas syringae, Pantoa agglomerans, Pantoa vagans, Paenibacillus wynnii, Staphylococcus caprae, Bifidobacterium asteroids, Staphylococcus caprae and Micrococcus luteus associatetd to Varroa destructor. Vanikova et al., 2015, recorded Microbacteruim sp and Bacillus sp. Because microorganisms are ubiquitous in nature, it is not surprising to find a variety of them associated with insects. Generally, the kinds of microorganisms involved with an insect reflect the microflora of the surrounding environment (Ingraham et al., 1975). The pathogenicity is largely associated with entry to the hemocoel either through a wound in the exoskeleton or more generally through the peritrophic membrane of the gut (Priest, 2000). Bacteria that fall within the category of insect pathogens, families characteristic of enthomopathogens, are: Bacillaceae, Enterobacteriaceae, Streptococaceae (Hrabak, 2003), Pseudomonadaceae, Lactobacillacea, micrococcaceae (Dhanasekaran and Thangar, 2014). It has been suggested that the presence of bacteria in the haemolymph is indicative of septicemia and a common sequelae to stress (Lightner, 1977in Gomez-Gil et al., 1998. According to Hubert et al., (2015), the mite could be reservoirs of the pathogenic bacteria in the apicultures.

CONCLUSIONS
Through our experience, apparently the heamolymph of the healthy emerging worker honeybee (Apis mellifera L) is free for bacteria. However, the bacterial contamination enable to be transmitted into honeybee heamocoel are: Bacillus licheniformis (4 strains), Bacillus mycoide (1strain), Bacillus coagulans (1strain), Brevibacillus chohinensis (1strain), Aeromonas hydrophila (1 strain) and Pantoa sp (1 strain). The knowledge of these bacteria microflora opens up a new perspective for integrated control of this parasite, which decimates bee colonies yearly.