A new report on Hesperomyces coleomegillae (Ascomycota, Laboulbeniales) parasitism of Coleomegilla maculata (Coleoptera, Coccinellidae) in Brazil

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.


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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 Production (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 integument were observed in the colony matrix at CIMP. Each individual was observed under a dissecting microscope in the Laboratory of Mycology (DEnF, UFRRJ). Examining 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 dissecting 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 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.

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 initiate 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 transmission of H. axyridis to Coccinella septempunctata L. and Olla v-nigrum (Mulsant), as well 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][33][34][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.