A new report on Hesperomyces coleomegillae (Ascomycota, Laboulbeniales) parasitism of Coleomegilla maculata (Coleoptera, Coccinellidae) in Brazil 1 of 7Published by Polish Botanical Society Acta Mycologica ORIGINAL RESEARCH PAPER A new report on Hesperomyces coleomegillae (Ascomycota, Laboulbeniales) parasitism of Coleomegilla maculata (Coleoptera, Coccinellidae) in Brazil Carlos Antonio Inácio1*, Hagabo Honorato de Paulo2, Jonas Dias Almeida2, Jessica Rembinski2, Elen Lima Aguiar Menezes2, Alessandra Carvalho Silva3 1 Department of Entomology and Plant Pathology, Universidade Federal Rural do Rio de Janeiro, BR 465, Km 7, University Campus, Rural Zone, Seropédica, RJ, CEP 23851-970, Brazil 2 Department of Entomology and Plant Pathology, Rural University of Rio de Janeiro, UFRRJ, BR 465, Km 7, University Campus, Rural Zone, Seropédica, RJ, CEP 23851-970, Brazil 3 Embrapa Agrobiology, BR 465, Km 7, Ecology Site, Rural Zone, Seropédica, RJ, CEP 23981-355, Brazil * Corresponding author. Email: inacio@ufrrj.br Abstract For the first time, the genus Hesperomyces has been reported to infect Coleomegilla maculata in laboratory mass rearing in Brazil. Thalli were found growing on several parts of this ladybird species, including the head, elytra, legs, and abdomen. Infested adults died after 60 days. Keywords host–parasitic interaction; Laboulbeniomycetes; ectoparasitic fungi; 12-spotted ladybird; biotrophic parasites; tropical fungi Introduction The order Laboulbeniales (Fungi: Ascomycota) comprises about 2,200 species in 141 genera [1] and was previously included in Basidiomycota and/or Zygomycota. Recent studies based on molecular data confirmed that Laboulbeniales are members of As- comycota [2]. They are biotrophic ectoparasites of arthropods, and beetles (Insecta: Coleoptera) and flies (Insecta: Diptera) are their most common hosts [3–5]. These fungi are specialized and develop by forming a multicellular thallus in the tegument of living arthropods [6–8]. Seven species of Laboulbeniales parasitize ladybirds (Coleoptera, Coccinellidae) [6]: six species of the genus Hesperomyces (H. chilomenes Thaxt., H. coccinelloides Thaxt., H. coleomegillae W. Rossi & A. Weir, H. papuanus T. Majewski & K. Sugiy., H. palustris W. Rossi & A. Weir, and H. virescens Thaxt.) and one Laboulbenia species (L. coccinellidicola Haelew.) [6,9]. An eighth species, H. hyperaspidis Thaxt., was brought into synonymy with H. virescens based on morphological characteristics [10], although this decision might have been premature [6]. Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae), the 12-spotted ladybird, is widely distributed in natural and managed ecosystems in North, Central, and South American regions, including Brazil [11–14]. It is primarily an aphidophagous insect, but its diet may include other insects (e.g., scales, psyllids, eggs, and neonate larvae of Lepidoptera and Coleoptera). Thus, this ladybird is considered an important agent for crop pest control [15–17]. Coleomegilla maculata adults are known to be hosts for H. coleomegillae and H. palustris in Ecuador, Costa Rica, and Cuba. The first observation of C. maculata acting as a host for Hesperomyces (H. palustris) occurred in 1951 in Cuba DOI: 10.5586/am.1117 Publication history Received: 2018-02-25 Accepted: 2018-11-21 Published: 2019-04-18 Handling editor Malgorzata Ruszkiewicz- Michalska, Institute for Agricultural and Forest Environment, Polish Academy of Sciences, Poland Authors’ contributions HH: collected and examined the material, drafted the manuscript; JD and JR: examined the material, prepared the figure; contributed to manuscript preparation; AS: wrote the manuscript; EM: wrote the manuscript and identified ladybird species; CA: examined and identified the material, wrote the manuscript Funding The authors acknowledge CAPES, CNPq, and FAPERJ for funds supporting this research. Competing interests No competing interests have been declared. Copyright notice © The Author(s) 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits redistribution, commercial and noncommercial, provided that the article is properly cited. Citation Inácio CA, de Paulo HH, Almeida JD, Rembinski J, Menezes ELA, Silva AC. A new report on Hesperomyces coleomegillae (Ascomycota, Laboulbeniales) parasitism of Coleomegilla maculata (Coleoptera, Coccinellidae) in Brazil. Acta Mycol. 2019;54(1):1117. https:// doi.org/10.5586/am.1117 Digital signature This PDF has been certified using digital signature with a trusted timestamp to assure its origin and integrity. A verification trust dialog appears on the PDF document when it is opened in a compatible PDF reader. Certificate properties provide further details such as certification time and a signing reason in case any alterations made to the final content. If the certificate is missing or invalid it is recommended to verify the article on the journal website. mailto:inacio%40ufrrj.br?subject=A%20new%20report%20on%20Hesperomyces%20coleomegillae%20%28Ascomycota%2C%20Laboulbeniales%29%20parasitism%20of%20Coleomegilla%20maculata%20%28Coleoptera%2C%20Coccinellidae%29%20in%20Brazil https://doi.org/10.5586/am.1117 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.5586/am.1117 https://doi.org/10.5586/am.1117 2 of 7© The Author(s) 2019 Published by Polish Botanical Society Acta Mycol 54(1):1117 Inácio et al. / A report on Hesperomyces parasitism of Coleomegilla maculata [9]. Coleomegilla maculata adults were found with H. palustris and H. coleomegillae thalli in Costa Rica and Ecuador [18]. The Brazilian Laboulbeniales fungi have not yet been thoroughly investigated. There is a record of Laboulbenia ecitonis Blum from the Eciton, or army ant, genus (Hymenoptera: Formicidae, Ecitoninae) in Curitiba, State of Paraná [7,19]. Data in previous literature indicates that nearly 100 species of laboulbenialean fungi have been recorded in Brazil, the majority of which have been found on beetles [20,21]. In addition, Rossi and Bergonzo [21] documented the occurrence of 13 Laboulbeniales species in Brazil – all but one associated with beetles. The exception was H. coccinelloides, found on a ladybird, Diomus seminulus (Mulsant), in the State of Ceará. This work aims to report Hesperomyces infecting C. maculata adults in mass-rearing laboratory conditions in Seropédica, Rio de Janeiro, Brazil. Material and methods A colony matrix of C. maculata was reared in a climate-controlled laboratory (25°C ±1°C, 60% ±10% RH, and 12-hour photoperiod) at the Integrate Pest Management Center (CIMP) of the Department of Entomology and Plant Pathology (DEnF) of the Universidade Federal Rural do Rio de Janeiro (UFRRJ) on the Seropédica campus in RJ, Brazil. This colony originated from adults collected from the organic farm Fazendinha Agroecológica Km 07, also known as Integrated System of Agroecological Produc- tion (SIPA) in the municipality of Seropédica, RJ (22°45'24" S, 43°40'29" W) in 2010 [22]. Adults and larvae were reared continuously and fed ad libitum on living larvae of Drosophila melanogaster Meigen (Diptera: Drosophilidae). The offspring of adults collected in this farm were introduced annually in the colony matrix to maintain the vigor of the colony. The field adults were separated from those of the colony matrix and held in plastic containers. Adults were stored in disposable 1-liter transparent plastic containers sealed with organza to enable gas exchange. Due to the difficulty in visually determining the sex [23], six adults were kept per container; however, when mating had not been observed in a 24-hour period, random exchanges among containers of some individuals were performed to ensure the presence of at least one viable pair per container. Filtered water was provided using cotton wool placed in plastic bottle caps. The larvae of C. maculata were individually kept in 20-mL glass vials closed with hydrophilic cotton from the second instar until adulthood. From September to December 2015 and January to May 2016, C. maculata adults (6 months to 1-year old) with yellowish structures in some parts of their integu- ment were observed in the colony matrix at CIMP. Each individual was observed under a dissecting microscope in the Laboratory of Mycology (DEnF, UFRRJ). Examin- ing these adults revealed that the visible structures were thalli of Laboulbeniales fungi. Their position on the host integument was recorded (Tab. 1). To identify the parasite, the thalli were gently removed from the host’s cuticle using a needle as well as dissect- ing and optical microscope techniques. The thalli were mounted on permanent slides stained with cotton-blue/ lactoglycerol or floxin/KOH glycerol. Measurements and pictures were taken using an Olympus BX41 optical microscope with a digital camera and micrometer. The following morphological characteristics were determined: total length from foot to perithecial tip, length from foot to tip of uppermost antheridium, length and width of perithecium, and length and width of ascospores. Both the Philco-Hitachi TM 1000 electron microscope, located at the Health and Sciences Biological Institute – ICBS/UFRRJ (Seropédica campus), and Evo LS 10 (Carl Zeiss), located at EMBRAPA Agrobiology (Seropédica, RJ), were Tab. 1 Number of mature thalli of H. colleomegillae on different body parts of several host individuals. Body part Number of thalli* % of thalli Head 25 8.9 Leg 44 16.0 Thorax 46 30.1 Abdomen 61 22.0 Elytra 64 23.0 Total 281 100.0 * Taken from 50 naturally infected individuals. 3 of 7© The Author(s) 2019 Published by Polish Botanical Society Acta Mycol 54(1):1117 Inácio et al. / A report on Hesperomyces parasitism of Coleomegilla maculata utilized for some measurements, and pictures were acquired. Identification of the fungal parasite followed de Kesel [24] and Goldmann et al. [18]. Sampled material and voucher slides were deposited at the Phytopathological Herbarium Verlande Duarte Silveira (DEnF/UFRRJ), UFRJ. Results The fungus was identified in the genus Hesperomyces, described by Thaxter in 1891 (Ascomycota, Laboulbeniomycetes, Laboulbeniales) [25]. Hesperomyces coleomegillae W. Rossi & A. Weir (Fig. 1) Material examined. Brazil, Rio de Janeiro, Seropédica: main campus of UFRRJ, inside a building of CIMP, coordinates 22°46'10" S, 43°41'38" W, on elytra of the ladybird Coleome- gilla maculata DeGeer, November 14, 2015, H. H. Paulo No. 3 (UFRRJ – 12.305). The fungus formed ascomata on several parts of C. maculata adults, including the prothorax, mesothorax, and head (antennae and mouthparts, such as palpi); however, formation primarily occurred on the elytra (dorsal part) (Fig. 1A–G). The fore, middle, and hind legs and abdomen also exhibited infection (Tab. 1). Ascomata showed the following characters: length from foot to top of perithecium: up to 770 μm; length from foot to tip of uppermost antheridium: 108–156 μm; perithecium: 92–563(–670) × 34–80(–96) μm, showing apical outgrowths (26–36 μm) (Fig. 1J–L); ascospores: 68–96 × 5–6 μm, hyaline, one-septate, spindle-shaped, with thick a mucilaginous layer (Fig. 1L) (Tab. 2). Discussion This paper reports the first record of living C. maculata adults hosting Hesperomyces in laboratory mass-rearing in Brazil, originating from specimens collected in the field. Eggs and larvae were never infected, which was expected as Laboulbeniales fungi only infect living adults [4,6]. This fungus was identified as H. coleomegillae based on morphological characters described in previous literature [18] (also see Tab. 2). It has already been reported that species in the genus Hesperomyces parasitize C. maculata adults; specifically, H. coleomegillae in Central America (Costa Rica) and H. palustris in Costa Rica, Cuba, and Ecuador [18,26]. Laboulbeniales fungi do not have a free-living stage, and the propagation of their sticky ascospores is triggered or promoted by the activity of the host (grooming, copulation, or other contact) [4,6,27]. A number of factors may have led the ladybird colony to become infected with H. coleomegillae. It is likely that a field-collected adult bearing mature thalli (having passed unnoticed by the collector) was enough to initi- ate propagation among the adults in the rearing containers. In addition, when mating had not been observed within 24 hours, there was random exchanges of individuals among the containers until the presence of at least one viable pair per container was observed. However, new adult collections should be carried out on the same farm to more accurately detect natural infection of C. maculata by Hesperomyces species. Individual Cycloneda sanguinea (Linnaeus), Eriopis connexa (Germar), Harmonia axyridis (Pallas), and Hippodamia convergens Guerin-Meneville adults have also been reported to occur at SIPA [13,28,29], and H. virescens Thaxt. has been reported to infect adult lady beetles of the above species in other countries [6,25]. Therefore, new records of species in Hesperomyces on wild populations of these lady beetles in Brazil should be expected. However, based on studies by Cottrell and Riddick [30] and Riddick [31] the chances of transmission taking place from H. virescens-infected H. axyridis to C. maculata adults at SIPA are likely low. These authors demonstrated that intraspecies transmission of this fungus in H. axyridis was common, whereas interspecies transmis- sion of H. axyridis to Coccinella septempunctata L. and Olla v-nigrum (Mulsant), as well 4 of 7© The Author(s) 2019 Published by Polish Botanical Society Acta Mycol 54(1):1117 Inácio et al. / A report on Hesperomyces parasitism of Coleomegilla maculata Fig. 1 (A–G) Body parts of Coleomegilla maculata infected by Hesperomyces. (A) Pronotum and dorsal part of the elytra of C. maculata with thalli (bar: 1 mm). (B,C) Legs and prosternum of C. maculata with ascomata (bar: 500 µm). (D–F) Pictures obtained from a scanning electron microscope (SEM). (D) Detail of the fungus on the head and pronotum of C. maculata (bar: 5 mm). (E) Infection on leg (bar: 600 µm). (F) Detail of infection of tibia (bar: 600 µm). (G) Detail of infection, mainly of femur (bar: 600 µm). (H–L) Hesperomyces structures. (H) Detail of young antheridium with appendages (bar: 15 µm). (I) Apical outgrowth of perithecia (bar: 100 µm). (J,K) Perithecia (bar: 100 µm). (L) Ascospores (bar: 20 µm). 5 of 7© The Author(s) 2019 Published by Polish Botanical Society Acta Mycol 54(1):1117 Inácio et al. / A report on Hesperomyces parasitism of Coleomegilla maculata as from O. v-nigrum to both C. septempunctata L. and H. convergens Guerin-Meneville, was notably uncommon. The same authors [30,31] did not observe infection of C. maculata adults after confinement with H. axyridis after placed in a vial and tumbled on a vial roller for 1 h. In general, Laboulbeniales fungi cause little or no harm to their arthropod hosts and do not seem to kill them [32–35]. However, there are indications that species in the genus Hesperomyces deviate from this because of the penetrating haustorium in the exoskeleton of their hosts, and some negative effects on hosts have been reported [36,37]. Hosts with high numbers of thalli, such as those from H. virescens on H. axyridis with more than 100 thalli (sometimes >400 thalli), on the outer parts of their body (e.g., elytra, eyes, antennae, mouthparts, and/or legs) may no longer be able to fly, mate, or detect their prey [6,37]. In this study, it was observed that 100% of naturally colonized adults died after 60 days and the number of eggs laid by infected adults was affected compared to noninfected adults of the same age. It was also observed that C. maculata adults under 6 months of age were not infected by H. coleomegillae. This result suggests that older adults may be more susceptible to infection by this species. Additional research is needed to support this hypothesis. Tab. 2 Comparison of morphological characters (in µm) of Hesperomyces species associated with Coccinellidae (Coleoptera) reported from Central and South America. Species Country Length from foot to perithecial tip Length from foot to tip of uppermost antheridium Length and width of perithecium Perithecial outgrowth Length and width of ascospores H. coccinelloides1 Panama 120–240 - 24–30 × 85–110 35–40 - H. coleomegillae2 Costa Rica, Ecuador 370–600 130–150 55–70 × 210–350 27–33 75–77 H. hyperaspidis3 Trinidad Tobago 180 - 25 × 110 20 - H. palustris2 Costa Rica, Ecuador (355)400–675 105–130 55–70 × 245–355 50–70 - H. coleomegillae4 Brazil Up to 770 108–156 34–80(–96) × 92–563(–670) 26–36 68–96 × 5–6 1 Thaxter [38] (host: Scymnus tardus Mulsant); 2 Goldmann et al. [19] (host: Coleomegilla maculata); 3 Thaxter [38] (host: Hyperaspis sp.); 4 present study (host: Coleomegilla maculata). Acknowledgments The second author acknowledges CAPES for grants supporting his master’s degree program. 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J Invertebr Pathol. 2007;94(3):196–203. https://doi.org/10.1016/j.jip.2006.11.002 38. Thaxter R. Contribution toward a monograph of the Laboulbeniaceae. Part V. Memoirs of the American Academy of Arts and Sciences. 1931;16(1):1–435. https://doi.org/10.2307/25058136 https://doi.org/10.2307/20020441 https://doi.org/10.1038/s41598-018-34319-5 https://doi.org/10.1590/S0102-05362010000100008 https://doi.org/10.2317/0304.18.1 https://doi.org/10.1155/2012/814378 https://doi.org/10.1673/031.006.4201 https://doi.org/10.1016/S0269-915X(09)80238-3 https://doi.org/10.1080/00275514.1996.12026705 https://doi.org/10.14411/eje.2008.049 https://doi.org/10.1016/j.biocontrol.2009.05.008 https://doi.org/10.1007/s10526-016-9766-8 https://doi.org/10.1016/j.jip.2006.11.002 https://doi.org/10.2307/25058136 Abstract Introduction Material and methods Results Hesperomyces coleomegillae W. Rossi & A. Weir Discussion Acknowledgments References 2019-04-18T12:46:43+0100 Piotr Otręba