Spore morphology and ultrastructure of an Ascosphaera apis strain from the honeybees (Apis mellifera) in southwest China

Abstract

Ascosphaera apis is an intestinally infective, spore-forming, filamentous fungus that infects honeybees and causes deadly chalkbrood disease. Although A. apis has been known for 60 y, little is known about the ultrastructure of the spores. In this study, the fine morphology and ultrastructure of an isolate, A. apis CQ1 from southwest China, was comprehensively identified by transmission electron microscopy, confocal laser scanning microscopy, scanning electron microscopy, and optical microscopy. The high sequence similarity and phylogenetic data based on nuc rDNA ITS1-5.8S-ITS2 (ITS) supported the hypothesis that the CQ1 strain is a new member of the A. apis species. Morphological observation indicated that the mature spores are long ovals with an average size of 2 × 1.2 µm and are tightly packed inside spherical spore balls. More than 10 spore balls that were 8-16 µm in diameter were wrapped and formed a spherical, nearly hyaline spore cyst of 50-60 µm in diameter. Ultrastructural analysis showed that mature spores have two nuclei with distinctly different sizes. A large nucleus with double nuclear membranes was found in the center of the spore, whereas the small nucleus was only one-fifth of the large nucleus volume and was located near the end of the spore. Numerous ribosomes filled the cytoplasm, and many mitochondria with well-defined structures were arranged along the inner spore wall. The spore wall consists of an electron-dense outer surface layer, an electron-lucent layer, and an inner plasma membrane. Chitin is the major component of the spore wall. The germinated spore was observed as an empty spore coat, whereas the protoplasts, including the nuclei, mitochondria, and ribosomes, had been discharged. In addition to these typical fungal spore organelles, an unknown electron-dense regular structure might be the growing mycelium, which was arranged close to the inner spore wall and almost covered the entire wall area.

The Effect of Artificial Media and Temperature on the Growth and Development of the Honey Bee Brood Pathogen Ascosphaera apis

“…In addition, the solitary bees’ brood is exposed to fluctuating temperatures, which could be the main reason for the greater spore cyst production. In former studies, smaller spore balls (12.5 µm, 12.0 µm, 12.1 µm) and bigger spore cysts (80.2 µm, 70.0 µm, 84.5 µm) were found at lower temperatures [ 62 , 64 , 73 ], while bigger spore balls (up to 16 µm) and smaller spore cysts (50–60 µm) were found at 30 °C [ 23 ]. The pattern can be seen in all the tested media in this study.…”

“…Ascospores, which are the only infective units causing chalkbrood [ 22 ], are formed in spore balls (asci) and located in resistant cysts [ 16 ]. The spores contain two nuclei; the bigger one lies in the center and the second smaller one is situated near the end of the spore [ 23 ]. The three-layered spore wall is tough, containing chitin as its major component [ 23 , 24 ], which helps ascospores stay viable for many years [ 20 , 25 , 26 ].…”

“…Chalkbrood is considered to be a stress-related disease [ 27 , 30 , 31 ], which could be the reason for its increased occurrence in recent years [ 22 , 23 ], because honey bees are stressed by many factors. According to Vojvodic et al [ 31 ], chalkbrood is a chronic disease because it persists in beehives for a long time and can break out at any time, depending on the conditions, which makes this disease more dangerous.…”

“…There are some indications that this fungus, which is one of the most contagious and destructive bee brood pathogens [ 14 ], may be increasing in occurrence [ 22 ]. There are also some locations, such as northern Thailand [ 39 ] and China [ 23 ], where this pathogen causes high bee mortality because the even less pathogen-susceptible Apis cerana cerana can be easily infected by A. apis [ 40 ]. This fact contributes to chalkbrood’s worldwide occurrence and adds to the importance of the disease.…”

Changes in the gene expression of chalkbrood resistance in Apis mellifera larvae infected by Ascosphaera apis

“…Chalkbrood has been extensively studied in terms of pathogen biology and host-pathogen interactions. Many studies, including morphology (Skou 1988;Li et al 2018), pathology (Aronstein et al 2007;Theantana and Chantawannakul 2008), and epidemiology (Flores et al 2005), on A. apis have been conducted. With the completion of the sequencing of A. apis , the comprehensive transcriptome analysis of A. apis was carried out, and recently 379 lncRNAs and 118 miRNAs were identified in the chalkbrood pathogen (Qin et al 2006;Cornman et al 2012;Guo et al 2018a, b).…”