Nosema disease in the honey (Apis mellifera L) infested with varroa mites in southern Spain Fj Orantes Bermejo, P García Fernández

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Fj Orantes Bermejo, P García Fernández. Nosema disease in the (Apis mellifera L) infested with varroa mites in southern Spain. Apidologie, Springer Verlag, 1997, 28 (3-4), pp.105-112. ￿hal- 00891409￿

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Nosema disease in the honey bee (Apis mellifera L) infested with varroa mites in southern Spain

FJ Orantes Bermejo P García Fernández

Dpto Producción , Centro de Investigación y Formación Agraria (CIFA), Camino de Purchil s/n, 18004 Granada, Spain

(Received 8 November 1996; accepted 7 May 1997)

Summary — Twenty-nine hives infested by Varroa jacobsoni were sampled over a 2-year period in order to find out their degree of infection by Nosema apis. The hives were situated in ten apiaries dis- tributed throughout southern Spain. N apis has been found in 90% of the apiaries sampled and in 55.17% of the hives studied, but only 5.1% of the were infected. We have found a low corre- lation between the average number of spores per infected bee in the positive samples and the percentage of infected bees (r2 = 0.2438; P < 0.001; n = 33), and between the average number spores in the composite samples of 60 bees and the percentage of infected bees (r2 = 0.4557; P < 0.001, n = 33). Our results show that N apis and V jacobsoni could develop independently and that those samples which manifested a low, medium and high infestation by V jacobsoni had percentage infections with N apis of 22.6% ± 3.6% vs 47.5% ± 16.2% vs 16.7% ± 10.4% respectively, without significant dif- ferences (F = 0.2817; P = 0.7567). A progressive increase in the number of spores per individual was detected with increasing levels of V jacobsoni infestation: 5.9 x 106 vs 9.1 ×6 10 vs 13.8 x 106 spores/bees, but no significant differences exist between them (F = 0.6053; P = 0.5531). Nosema apis / infection level / Varroa jacobsoni / Spain

INTRODUCTION effects include the loss of adult bees in win- ter and a reduction in the hive’s productivi- ty (Farrar, 1947; L’Arrivée, 1966; Cantwell The microsporidian Nosema apis Zander is and Shimanuki, 1969; Fries et al, 1984). an endoparasitic protozoan of honey bees Some of these losses may be related to the which causes considerable economic losses three viruses associated with N apis (Bai- in the beekeeping industry. Detrimental ley and Ball, 1991).

* Correspondence and reprints Tel: (34) 58 26 73 11; fax: (34) 58 25 85 10; e-mail: [email protected] or: [email protected] Nosema apis is widely distributed MATERIALS AND METHODS throughout the world (Bradbear, 1988), but whilst its effects are not considered impor- During the period October 1990 to October 1992, tant in tropical and subtropical climates samples were taken monthly from 40 Layens beehives distributed 12 in the (Wilson and Nunamaker, 1983), this is not among apiaries south of the Iberian peninsula, to ascertain the the case in countries with cli- temperate dynamics of the V jacobsoni population (Orantes mates. Differences in the seasonal incidence Bermejo et al, 1994; García Fernández et al, of infection depending on geographical loca- 1995). One hundred and sixty-two (162) sam- tion are well documented (Bailey and Ball, ples of bees from 29 of 40 beehives belonging 1991; Fries, 1993). to ten apiaries were analysed, to evaluate the extent of N apis infection. In Spain, N apis is well known, having Adult worker bees were collected from brood from mixed brood nests and stor- been detected by many researchers. How- nests, honey age areas, and a few were taken from the hive ever, almost all the published studies have entrance. This provided us with a good repre- been limited to the of the citing presence sentation of the hive’s population (Calderone microsporidia (Torrens Pastor, 1947; Habela and Shimanuki, 1992) and minimised the effects et al, 1987; Gijón-Botella et al, 1987), occurring as a result of the infected bees found in whereas hardly any studies have been made particular locations within the hive (Moeller, of its seasonal incidence. Pajuelo and Fer- 1956; L’Arrivée, 1963; Pickard and El-Shemy, nandez Arroyo (1979) report N apis infec- 1989). The bees, whose age was unknown, were conserved in 70% ethanol. tion levels of 21 % (number of samples and locations unknown), being most prevalent Sixty bees were individually analysed from each The abdomen of each bee was dis- in the months of January to April. sample. sected and placed in an Eppendorf tube, homogenising it with a small piston in distilled The disease evolves without producing water with a drop of 10% Nigrosin solution, any visible signs, meaning that in many finally adjusting the volume to 1 mL. A drop of cases no treatment is given. Other factors, this solution was placed in a haemocytometer to a of 0.1 mm under such as mite infestation, with a consequent give liquid depth small grid areas of 0.0625 mm2. The number of in 20 weakening of the colony, may influence the spores was counted, the total number subsequent development of N apis infec- grid squares being calculated by the expression: number of spores = tion levels. 8000× f; where f is the dilution factor used. This method is considered optimum for this calcula- The infection of bees by this protozoan of tion either for individuals or groups (Burnside colonies infested with the Varroa jacobsoni and Revell, 1948; L’Arrivée, 1963; Doull 1961; Oudemans mite is poorly documented. Cantwell, 1970; van Laere et al, 1980; Wilson and Nunamaker, 1983; Pickard and Smirnov (1978) suggests that the mortality El-Shemy, 1989) and its accuracy has been by rate in these circumstances increases investigated rapidly van Laere et al ( 1980). if no action is taken. In a made in study Various mean were made Romaniuk and analyses (Anova Poland, Wawrzyniak (1991) and Manova) and correlations between different concluded that the two coexisted. parasites parameters carried out. In order to make the vari- ance homogenous in these mean analyses, the The aim of this research is to find out data were transformed by the expressions for both the whether there is an interrelationship between y=Ln(x) average number of spores infected bee in the and the these two pathologies in Spain. The preva- per positive samples average number of spores in the composite sam- lence and distribution of N apis in our ples of 60 bees; and y = arctag(x) for the per- distributed experimental apiaries, through- centage of infected bees in the positive samples. out the southern half of the Iberian penin- For all calculations we used SPSS. (Nie et al, sula, are also reported. 1983). RESULTS the greater the number of spores, yet no sig- nificant differences existed in the means of Nosema apis in southern Spain the three different V jacobsoni-infested groups (F = 0.6 053; P = 0.5531) nor was there a correlation between the Table I sets out the results of the sampling. significant N apis is widely distributed, being diag- two parameters (percentage of parasitiza- nosed in 90% of the apiaries, with 55.17% of tion by V jacobsoni / number of N apis P = the colonies monitored in our study being spores; (r2 = 0.2 946; 0.102). infected. In total, 9720 bees were analysed, of which 495 were infected, which repre- sents 5.1 %. These infected bees contained DISCUSSION a mean of 7.6 x 106 ± 1.7 x 106 spores per bee (mean ± SE). N apis is an important pathogenic agent of hives which does not to have much As can be seen in table I, we found that N appear effect in our climatic apis is most prevalent during spring, and in conditions, causing are and the colder months from November to Jan- losses which undetectable insignif- icant to % of bees uary, except in the high mountainous regions beekeepers (5.1 analysed). (Lanjarón and Vadillo-Cazorla) where this However, it has a fairly high prevalence occurred earlier, in September. (55.17% of hives) and some bees develop large numbers of spores in their intestines In our we have found a experiment, sig- (the maximum found in this study = 86.4 x nificant relationship between the average 106 spores/bee). number of spores per infected bee in the positive samples and the percentage of We found that 20.37% of samples were infected = a simi- infected bees, but with a low coefficient of by N apis (n 162), figure lar to the results of a of sam- correlation (r2 = 0.2438; P = 0.003; n = 33) previous study from where it was found to be and between the average number of spores ples Spain in the composite samples of 60 bees and the 21% (Pajuelo and Fernández Arroyo, 1979). The was made to the percentage of infected bees (r2 = 0.4557; previous study prior P<0.001, n = 33). arrival of V jacobsoni on the Iberian Penin- sula. This fact, together with the results set out in table III, lead us to believe that the two coexist but N apis versus V jacobsoni parasites develop indepen- dently of one another. Table II shows the comparison between the The hypothesis that a weakening of the number of samples infected by N apis and colony caused by V jacobsoni infestation those infested with V jacobsoni: in 18.52% could alter internal factors in the bee, such as of the samples (n = 162) both parasites were the permeability of the middle intestine found. It can be seen from table III how walls, the effect of pteridines and the role similar positive percentages were found in of the intestinal microflora (Hartwig, 1976; colonies with both low and high infestations Peroutka and Cihar, 1978), and thus pro- by V jacobsoni, 22.8% vs 26.3%. Similarly voke a greater multiplication of N apis, is there are no significant differences in the still a matter of speculation. In this study, percentage of infected bees in the positive the colonies with the highest range of infes- samples 22.6% ± 3.6% vs 47.5% ± 16.2% vs tation by V jacobsoni had more spores per 16.7% ± 10.4% (F = 0.2 817; P = 0.7 567). individual bee but, as stated in the results, However, it was noted that the higher the there were no significant differences percentage of parasitization by V jacobsoni, between the means of these three groups

with different ranges of infestation by V number of haemocytes (Smimov, 1978) and jacobsoni. a blockage in the metabolism of proteins (Domatskaya, 1980), which may lead to a Similar results were obtained in a study decrease in the resistance of the honey bee carried out in Poland (Romaniuk and to N apis, as multiplication of N apis is Wawrzyniak, 1991) which point to a syn- favoured by the destruction of hemocytes ergic effect between V jacobsoni and N apis. (Gilliam and Shimanuki, 1967) and the Sixty-one samples were found to be infected acceleration of the catabolism of fats by N apis out of 209 samples with parasiti- (Gontarski, 1952). Gontarski (1952) reported zation from 0.1%- by V jacobsoni ranging a relationship between the reserves of fats 5% and a mean number of spores of 8.9 x and proteins in the honey bee and its resis- whereas in 20 with 106, samples parasitiza- tance to N apis. tion by V jacobsoni ranging from 25.1%- 100%, they found six samples infected with Under the conditions of our experiment, we found correlations between N apis, and the mean number of spores went significant the number of infected up to between 1.8 and 27.2 x 106. average spores per bee in the positive samples and the per- The parasitic action of V jacobsoni on centage of infected bees, but with a low the honey bee causes a loss in the volume of coefficient of correlation (r2 = 0.2438; haemolymph (Tewarson, 1983), a fall in the P=0.003; n = 33), the same occurs between the variables, average number of spores in Résumé — La nosémose chez les abeilles the composite samples of 60 bees and the (Apis mellifera L) infestées par l’acarien percentage of infected bees (r2 = 0.4 557; Varroa dans le sud de l’Espagne. Pour P<0.001, n = 33), contradicting the find- connaître l’incidence de Nosema apis, nous ings of L’Arrivée (1963), Bailey (1968), avons étudié 29 ruches infestées par l’aca- Flinger et al (1982) and Fries et al (1984). rien Varroa jacobsoni et ayant un taux d’infestation des abeilles adultes connu. With regard to the seasonality of N apis, L’échantillonnage a été réalisé d’octobre 5.1 % of bees were found to be only posi- 1990 à octobre 1992, dans dix ruchers répar- tive, it difficult to the data making represent tis dans le sud de l’Espagne (fig 1). Soixante or in the various climatic compare averages abeilles ont été analysées individuellement regions in which the hives were situated. par échantillon. L’abdomen de chaque We can only conclude that N apis is most abeille a été disséqué et mis à macérer dans prevalent during spring (May-July), except de l’eau distillée additionnée d’une de in mountainous goutte high regions (September). solution de Nigrosine à 10 % ; le volume Our station with the mildest climate (Maro, final a été ajusté à 1 mL. Les spores ont été with a Mediterranean subtropical climate), dénombrées à l’aide d’un N where and nectar exist hémocytomètre. plentiful pollen est dans toute la the is where the lowest apis largement répandu throughout year, étudiée et dans 90 % des rates and fewest in the région présent parasitization spores ruchers échantillonnés et dans 55.17 % des bees analysed were found, probably due to ruches étudiées. Son incidence est pourtant the fact that the bees which developed nose- faible : 5.1 % = des abeilles ana- mosis defecate and die outside the hive. (n 9720) lysées (tableau I). N apis présente une varia- A series of circumstances exist which tion saisonnière avec deux maximums, de may mean that this study would offer dif- mai à juillet et de novembre à janvier, sauf ferent results if it were repeated. For exam- dans les ruches situées en montagne où le ple, during the sampling period, Spain was deuxième maximum commence en sep- suffering a severe drought, which had con- tembre. Nous avons trouvé une relation sequential limiting effects on flowering. significative entre les paramètres suivants : Climatic conditions are important and have nombre moyen de spores par abeille infec- meant that many studies carried out in the tée dans les échantillons positifs et pour- same region give different results (Bailey centage d’abeilles infectées, mais avec un and Ball, 1991). faible coefficient de corrélation (r2 = 0.2438 ; P = 0.003 ; n=33). Cette relation est éga- lement valable pour le nombre moyen de d’abeilles infectées dans les échan- ACKNOWLEDGMENTS spores tillons composés de 60 abeilles et le pour- centage d’abeilles infectées (r2 = 0.4557 ; Our most sincere thanks to BV Ball (Rotham- P < 0.001, n = 33), ce qui contredit les résul- sted Experimental Station) for her comments on tats des études précédentes. Les deux para- some of the critical points in this research, and to sites ont été trouvés F Torres Ruiz (Statistic Department, Faculty of dans 18,52 % des Science, University of Granada) for his assis- échantillons (n = 162) (tableau II). Nos tance in the statistical data processing. résultats montrent que V jacobsoni et N apis peuvent se développer indépendamment l’un This research was backed the INIA by pro- de l’autre. Les échantillons faiblement, ject No 95073. We would like to give our special et hautement infestés thanks to I Martin Bravo and A González Mejías moyennement par for their assistance in the laborious spore-counts Varroa présentaient un taux d’infestation of N apis. par N apis de 22.6 % ± 3.6 %, 47.5 % ± 16.2 % et 16.7 % ± 10.4 % respectivement, pro infizierter Biene in positiven Proben sans différence significative entre ces trois und dem Prozentsatz von infizierten Bie- moyennes (F = 0.2817 ; p = 0.7 567) nen. Allerdings ist der Korrelationkoeffei- (tableau III). Une augmentation progressive zient mit r2 = 0,2438; p < 0,001; n = 33) du nombre de spores/individu a été observée klein. Eine ähnliche Beziehung bestand zwi- parallèlement à l’augmentation du taux schen den Variablen: Mittelwert der Spo- d’infestation par V jacobsoni (5,9 x 106; renanzahl in den aus 60 Bienen zusammen- 9,1 x 106 ; 13,8 x 106 spores/abeille res- gesetzten Proben und dem Prozentsatz der pectivement pour un taux d’infestation infizierten Bienen.(r2 = 0,4557; p < 0,001; faible, moyen et élevé), mais les différences n = 33), was den früheren Befunden wider- ne sont pas significatives (tableau III). spricht. In 18,52% der Proben (N = 162) wurden beide Parasiten, V jacobsoni und Nosema apis / taux infestation / Varroa N apis nachgewiesen (Tabelle II). Unsere jacobsoni / Espagne Ergebnisse zeigen, daß sich N apis und V jacobsoni unabhängig voneinander ent- wickeln und daß die Proben, die einen nied- mittleren oder hohen Befall durch V Zusammenfassung — Nosematose bei rigen, aufwiesen zu bzw L) bei Befall jacobsoni 22,6% ± 3,6% Honigbienen (Apis mellifera bzw mit N mit Varroa - Milben in Südspanien. Aus 47,5% ± 16,2% 16,7% ± 10,4% infiziert waren. Es demnach keinen 29 Bienenvölkern mit einer bekannten apis gibt Unterschied = = Befallsrate von Oud wur- signifikanten (F 0,2817; p Varroa jacobsoni wurde mit den während einer Periode von 2 Jahren an 0,7567) (Tabelle III). Allerdings einem in der Anzahl verschiedenen des Jahres adulte progressiven Anstieg Zeitpunkten der Einzelbiene auch ein stei- Bienen entnommen und der Befallsgrad mit Sporen pro von fest- Nosema apis bestimmt. Insgesamt wurden gender Befallsgrad V jacobsoni x 106 / Biene bzw x 162 Bienenproben gesammelt. Die Bienen- gestelt (5,9 Sporen 9,1 106 bzw 13,8 x 106 Sporen / völker waren auf 10 Bienenständen in ganz Sporen / Biene aber diese Unterschiede waren nicht Südspanien verteilt (Tabelle I). Von jeder Biene), = = Probe wurden 60 Bienen entnommen und signifikant (F 0,6053f; p 0,5531) einzeln untersucht. Hierzu wurden zunächst (Tabelle III). die Abdomen in destilliertem Wasser mit Nosema Zusatz von einem Tropfen 10% Nigrosin- apis, Infektionsgrad, Varroa jac- lösung zerquetscht, das Volumen dann auf 1 obsoni Oud, Spanien mL justiert und die Sporen mit einem Hae- mocytometer (Zählkammer) ausgezählt. N apis ist in der untersuchten Gegend weit REFERENCES verbreitet und wurde in 90% der Bienen- stände bei 55,2% der Bienenvölker nach- Bailey L (1968) The measurement and interralation- ships of infections with Nosema apis and M mel- gewiesen. Die Infektionsrate war mit 5, 1% lificae of honeybee population. J InvertebrPathol (n = 9720) befallenen Bienen jedoch gering 12, 175-179 (Tabelle 1). 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