doi:10.12741/ebrasilis.v7i3.449 e-ISSN 1983-0572 Publicação do Projeto Entomologistas do Brasil www.ebras.bio.br Distribuído através da Creative Commons Licence v3.0 (BY-NC-ND) Copyright © EntomoBrasilis Copyright © do(s) Autor(es)

The Metapleural Secretion of laticeps (Forel) does not have Fungicide Effect on the Entomopathogenic Fungus Beauveria bassiana (Bals.) Vuill. Luciane Kern Junqueira¹ & Elena Diehl²

1. Pontifícia Universidade Católica de Campinas, e-mail: [email protected] (Autor para correspondência). 2. Sem vinculação institucional, e-mail: [email protected]. ______

EntomoBrasilis 7 (3): 207-210 (2014)

Abstract. The metapleural gland (MPG) is an exocrine structure, exclusive to formicids, being more developed in females and workers. This structure is stunted or nonexistent in males. Several possible functions for the secretion produced by this gland have been suggested, but a protection against entomopathogens is the most accepted hypothesis. To test this, we applied a suspension of spores (1.30 x 106) of Beauveria bassiana (Bals.) Vuill. on the MPG and the antero-superior portion of the metathorax (ASM) of winged females of Acromyrmex laticeps (Forel). We only applied the suspension in the ASM of males, since they do not have the atrium characteristic in the MPG. Our results indicated that the metapleural secretion of females has no fungicidal effect on B. bassiana, and therefore both males and winged females are sensitive to this entomopathogenic fungus.

Keywords: Attini; entomopathogenic fungus; intracolonial assepsy.

Ausência de Efeito Fungicida da Secreção Metapleural de Acromyrmex laticeps (Forel) Contra Beauveria bassiana (Bals.) Vuill.

Resumo. A glândula metapleural (GMP) é uma estrutura exógena, encontrada apenas nos formicídeos, sendo mais desenvolvida nas fêmeas e operárias, enquanto nos machos ou está atrofiada ou não existe. Tem sido sugerido várias possíveis funções para a secreção produzida por esta glândula, sendo a hipótese de efeito protetor contra entomopatógenos a mais estudada. Para testar esta hipótese, foi aplicada uma suspensão de conídios (1,30 x 106) de Beauveria bassiana (Bals.) Vuill. nas GMP e na porção ântero-superior do metatórax (ASM) de fêmeas aladas de Acromyrmex laticeps (Forel), enquanto nos machos a suspensão foi aplicada apenas na porção ASM, pois estes não apresentam o átrio característico da GMP. Os resultados obtidos indicaram que a secreção metapleural das fêmeas desta espécie não possui efeito fungicida sobre B. bassiana, e que tanto os machos como as fêmeas alados são sensíveis a este fungo entomopatogênico.

Palavras-chave: Assepsia intracolônia; Attini; fungo entomopatogênico.

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nts have many natural enemies, including viruses (Va l l e s However, only the last two have not receive severe critics (Ye k & et al. 2005), bacteria (Pi n t o et al. 2003; De d e i n e et al. Mu e l l e r 2010). Entomologia Geral 2005), microparasites and parasitoids (Sc h m i d t -He m p e l 1998; Br o w n 1999), invertebrates (To n h a s c a et al. 2001; Ro l f f The function of the MPG has been subjected to study since the & Si v a -Jo t h y 2003), and vertebrates (Ja f f é 1993; Di e h l -Fl e i g 1970s, but the results are often conflicting, especially involving 1995a; 1995b). Accordingly, they also display numerous defense a possible antiseptic effect of its secretion (Ye k & Mu e l l e r mechanisms, including behavior (Di e h l -Fl e i g & Lu c c h e s e 1991; 2010). Antibiotic substances produced by MPG and possibly Ja f f é 1993), increased genetic diversity, either by polyandry or other glands have an essential significance for fungus-growing polygyny (Sc h m i d -He m p e l 1994; Cr o z i e r & Fj e r d i n g s t a d 2001) (: Attini), since these ants must protect the immunological mechanisms (Sc h m i d -He m p e l 2005), especially symbiotic fungus which they cultivate and feed upon, besides the secretions of metapleural glands (Hö l l d o b l e r & Wi l s o n 1990; maintenance of the assepsy of the ants themselves. This secretion Sc h m i d -He m p e l 1998; Sc h l ü n s & Cr o z i e r 2009). Despite these has also been suggested as responsible for the characteristic odor defense mechanisms, the occurrence of entomopathogenic fungi of each colony required for the recognition of its individuals, in ants is relatively common (Pe r e i r a & St i m a c 1992; Di e h l -Fl e i g but the absence of this secretion in males may indicate that this et al. 1993; Oi et al. 1994; Di e h l & Ju n q u e i r a 2001). behavior may be mediated by other odor sources (Hö l l d o b l e r & En g e l -Si e g e l 1984). Formicids are the only that share a unique defense mechanism consisting of a pair of exocrine glands located in the Here, we tested if the metapleural secretion of reproductive posterolateral portion at the end of the mesosome (Bo t et al. 2002). individuals of fungus-growing ants (Myrmicinae: Attini) have Such glands are rare in males and arboreal (Hö l l d o b l e r fungicidal effect on the entomopathogenic fungus Beauveria & Wi l s o n 1990; Ja f f é 1993). Although not mutually exclusive, bassiana (Bals.) Vuill.. For this, we used both females and winged four hypotheses about the function of these metapleural glands males of Acromyrmex laticeps (Forel) collected soon after two (MPG) have been proposed, it would function as: 1) intracolonial nuptial swarming to: 1) determine the frequency of individuals or intraspecific recognition, 2) determinant of territory either/ or nest entrance, 3) chemical defense, or 4) antiseptic action. Funding Agency: FAPERGS. www.periodico.ebras.bio.br 208 Table 1: Number of winged males and females of frequency ofinfectedindividuals. individuals of the same colony had already been reported (S in infection to susceptibility differential The 1). (Table reservoirs metapleural the in secretion a contained females of 91.7% Thus, reservoir. right the only in secretion had (0.84%) individual one with metapleural secretion in 109 individuals (90.83%), and only and specifically in the case of formicids, antiseptic substances produced antiseptic by MPG offormicids, (B case the in specifically and L in wingedfemalesof and (H kinship parasites intracolonial and to vulnerable particularly pathogens due to are high individual density, high levels insects of interaction Social of females 120 and males 20 with resistance We conducted a bioassay to determine sex differences in pathogen to swarm, same the evaluate theoccurrenceofMPGunderstereomicroscope(40x). from males 35 collected also We female. each of gland right or left either in reservoir the in secretion a of presence the recorded We (40x). stereomicroscope under needle the was wedrilled female each micro with portion upper-middle Then, their in reservoirs metapleural wings, tweezers. fine-tipped the with of immobilized removal the After early summer. in flight nuptial a after females winged 109 collected We that pathogen. to resistance the in differences sex possible investigate 2) and reservoirs its in secretion a containing MPG of pair a with D (H H & S (D ants Attini in length in more studied 1990). The effects of antiseptics metapleural secretion have been 1993; each groupwerecomparedusingachi-squaredtest( dead and infected individuals daily during 15 days. The results in and a 12:12 hours light: dark regime. We recorded the number of Jars solution. remained in a sterile, sugar moist chamber at 27±1 aqueous ºC, at 80% 25% RH, of mL 0.05 in soaked bottom the at paper filter of disk a with jars, sterilized in remained Each equal numberofmales. As a control, we applied a sterile saline solution to the ASM in an have males. 10 not of ASM the do to only applied males was suspension the Since MPG, females. other 40 in solution saline sterile a applied we control As chamber. flow laminar sterile a in females 40 another of ASM the in and females 40 of MPG the in eair hgee f oh niiul ad et (D nest and individuals both of hygiene behavior, defense mechanisms to deal with this situation, such as avoidance e on ta te P rsror o tn 83% ot f 120 of out (8.33%) of ten females of reservoirs MPG the that found We 2002; f niiul ta my fet mue ucin (R function immune affect may that individuals of of suspension a of consisted test This flight. nuptial another after Total Metapleural gland Control Antero-superior portionofthemetathorax e s e h c c u The MetapleuralSecretionof o m e s e g g u B. bassiana B. N c a m i t p l e o t n e m i c s a B B 1994) and could explain the differences of infection found t o r e a B & 1992; 1991;

et al. et

. laticeps A. 2005) and in some in and 2005) al. et a m s m o o Results andDiscssion Application region Material andethods (1.30 x 10 x (1.30

O 2002). These insects have developed numerous developed have insects These 2002). 1996; al. et F i etal.1994; h s t i r n e l l i 04 B 2004; A. laticeps. D & ee mt, oh eevis ee filled were reservoirs both empty, were & V 6 ) being applied with a microcapillary a with applied being ) Acromyrmexlaticeps l h e i D n a B r e a l h e i l l ö e n i t x e B -F o

d J & g i e l v t al. et o n e b r e l &T Solenopsis 1987; 96, mue defenses immune 1996), a r i e u q n u 05, g ad gender and age 2005), & r o h l h e i Acromyrmex laticeps, local application of a conidia solution (1.30 x 106) of H W A. laticeps A. v l l ö -F n o s l i n o s l i 2001; (Forel)… g i e l species (P species d Tested winged o 2002). b 1990

r e l l h e i Z t al. et f f l o females r a B & 120 collected -F 40 40 40 t o 1999).

; W 2001), i m h c 1993; g i e l

a r i e r e t al. et J n o s l i é f f a & d -

V very small, with the atrium closed or absent in males (H is it while workers, than females in developed more is gland this subgenus to ants, parasitic hypertrophied in of species that use it species as a defense, several such those in the in absent or reduced from control winged females (χ detected We of infection. difference (χ difference bassiana while infected, not was ASM the in inoculated male one Only akn mtperl lns (J glands metapleural lacking males with workers, the than larger are subfamilies Ecitoninae cie n h cln ad hi esnil ucin s fertilization (H is function essential their and colony the in active (χ significant highly are differences the control, females the of that with infection of frequencies the these compare we than when But MPG ASM. the in inoculated were females when the pathogen to resistance higher provide not did secretion metapleural hn hs dt ae oprd ih ht band n h male the control in group the differences are not obtained significant (χ that with compared are date these when ln. hs s h ms pcla pr o ti gad (B gland this of part peculiar metapleural most the the is to This externally gland. atrium characteristic the have eotd ht ae o sm seis sc as such species, some allardycei of males that reported H structure wouldbeuseless. specific such and females fertilizing after die they since laticeps, males in MPG the expected not would we together, features & this gland are unknown (Y unknown possess are males gland this only which in species and males, in common is h piay ucin acietr, n rltv sz o MPG of size (Y vary may relative and architecture, function, primary The a contaminationduringthebioassays(Table1). hy ol nt rdc te erto wt psil antiseptic (B possible function with secretion the produce not would they MPG, lack males since However, group. males the of size sample small our by biased been have could results these However NS). was difference this However, 1). (χ significant not (Table when 85% ASM and the MPG to the applied in inoculated was fungus the when by infection of frequency The This situation is similar in immune deficient vertebrates when vertebrates deficient immune in similar is situation fungus. This non-pathogenic the of influence negative the allowing indicated that the pathogenic fungus broke the resistance of ants, flavus Sorokin the entomopathogenic fungus B e a aray on peiul ta mls of males piriventris that previously found already had We Iridomyrmex purpureus perpilosa r e a n a s e g g u 2 frequency (%)of infected females E l l ö = 44.252,df3,P<0.0001). B l e g n Number and

et al. et d o (Link) o 32(80.5) 29(72.5) & B v

5(12.5) b n e -S r e l n te potnsi, vrln fungus avirulent opportunistic, the and was leaving the joints in 90% of them (Table 1). This 1). (Table them of 90% in joints the leaving was Physocrema l e g e i 97. h fmls n mls f h Drlne and Dorylinae the of males and females The 1987). (Wheeler), 2005). However, 2005). (Santschi) r e a (Mann), a m s m o o k e & 2

in = 31.0, df = 1, P< 0.001) was highlysignificant, but . bassiana B. & M

1984; et al. et Acromyrmex echinatior W 2 = 3.00, df = 1, NS), which indicates that the that indicates which NS), 1, = df 3.00, = n o s l i (2004) investigated the interaction between interaction the investigated (2004) r e l l e u ooesr cockerelli Novomessor 2005), thus explaining the high frequency high the explaining thus 2005), ymccsu mimicus Myrmecocystus of the the of and H 90 D 1990; l l ö (Smith), havetheseglands. 2 Tested Males 2010). The size of this gland can vary can gland this of size The 2010). = 1.0, df = 3, P < 0.01), possibly due to n 0 o cnrl ae ad 12.5% and males control of 10% in k e coymx heyeri Acromyrmex d o Metarhizium anisopliae & H b B. bassiana B. é f f a 20 r e l 10 10 l l ö -- l h e i M & d 93. h asne f MPG of absence The 1993). r e l l e u o Crematogaster -F b r e l W g i e l n o s l i (Wheeler). Their results e-ISSN 1983-0572

& 2010). Males are little are Males 2010). in females was 72.5% was females in 95) Tkn these Taken 1995b). Beauveria bassiana E Junqueira &Diehl 90 J 1990; frequency (%)of l e g n (André), infected males Number and Frl dd not did (Forel) Welr and (Wheeler) 2 -S 9(90.0) = 2.22, df = 1, 1(10.0) Temnothorax Atta . Meanwhile, . l e g e i Aspergillus -- (Metschn.) é f f a l l ö Formica sexdens n e l l i (1984) d 1993; o b

and r e l & A. B.

Setembro - Dezembro 2014 - www.periodico.ebras.bio.br EntomoBrasilis 7 (3) infected with an opportunistic parasite. piriventris Santschi (Myrmicinae, Attini), and its effect on the entomopathogenic fungus Beauveria bassiana (Bals.) A previous study (Ba e r et al. 2005) compared the immune Vuill. Neotropical Entomology, 30: 517-522. responses of workers and males of A. echinatior to a virulent Diehl-Fleig, E. & M.E. de P. Lucchese, 1991. Reações parasite. Since males had lower immune responses than workers comportamentais de operárias de Acromyrmex striatus have, the authors suggested that this difference could be due (, Formicidae) na presença de fungos to the lower genetic diversity due to haploidy or the reduced entomopatogênicos. Revista Brasileira de Entomologia, 35: investment in immunity by males. It could also be due to the 101-107. fact that the diploid workers have higher and more genetic Diehl-Fleig, E., 1995a. Sucesso no estabelecimento de colônias de diversity, and variable ages, since they are continually produced. Acromyrmex striatus (Roger) (Hymenoptera, Formicidae). Conversely, males are born only in the reproductive period, and Anais da Sociedade Entomológica do Brasil, 24: 625-630. would have virtually no variation in age. Nonetheless, when Diehl-Fleig, E., 1995b. Formigas: organização social e ecologia workers were exposed to M. anisopliae, they showed a reduced comportamental. Ed. Unisinos, São Leopoldo, 168p. immune response after four days, suggesting that the immune Diehl-Fleig, E., M.E. da Silva, A. Specht & M.E.V. Labres, 1993. system was destroyed by the virulent parasite. Efficiency of Beauveria bassiana for Acromyrmex spp. control (Hymenoptera: Formicidae). Anais da Sociedade In this study, the high frequency of males and winged females Entomológica do Brasil, 22: 281-285. infected with B. bassiana could be due to the metapleural Do Nascimento, R.E., E. Schöesters, E.D. Morgan, J. Billen & D.J. secretion that has reduced fungicidal effect, but also to an Stradling, 1996. Chemistry of metapleural gland secretions of inefficient immune system (Ba e r et al. 2005). The contamination three attine ants, Atta sexdens rubropilosa, Atta cephalotes, in our control group somewhat reinforces this hypothesis. and Acromyrmex octospinosus (Hymenoptera: Formicidae). Our results indicate that the metapleural secretion of winged Journal of Chemical Ecology, 22: 987-1000. females of A. laticeps do not have an effective fungicidal effect Fritsh, S. & E. Diehl-Fleig, 1996. Reações comportamentais de on B. bassiana. However, we suggest that other chemical and Acromyrmex heyeri e A. striatus (Hymenoptera - Formicidae) immunological mechanisms associated or not with behavioral à fungos filamentosos. 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Suggested citation:

Junqueira, L.K. & E. Diehl, 2014. The Metapleural Secretion of Acromyrmex laticeps (Forel) does not have Fungicide Effect on the Entomopathogenic Fungus Beauveria bassiana (Bals.) Vuill. EntomoBrasilis, 7 (3): 207-210. Available in: doi:10.12741/ebrasilis.v7i3.449 210

e-ISSN 1983-0572