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Article

Fungal Flora in Adult Females of the Rearing Population of Ambrosia Beetle Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae): Does It Differ from the Wild Population?

by
Zi-Ru Jiang
1,
Hayato Masuya
2 and
Hisashi Kajimura
1,*
1
Laboratory of Forest Protection, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
2
Department of Forest Microbiology, Forestry & Forest Products Research Institute (FFPRI), Tsukuba 305-8687, Japan
*
Author to whom correspondence should be addressed.
Diversity 2022, 14(7), 535; https://doi.org/10.3390/d14070535
Submission received: 26 May 2022 / Revised: 28 June 2022 / Accepted: 28 June 2022 / Published: 30 June 2022
(This article belongs to the Topic Fungal Diversity)
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Abstract

:
Ambrosia beetles bore into host trees, and live with fungi symbiotically that serve as a food source. However, it is challenging to directly observe these beetles in the wild. In this study, Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae), a pest of fig trees in Japan, were reared under artificial conditions to emulate the behavior of ambrosia beetle. Fungi were isolated from the adult females of E. interjectus to identify the species associated with secondary symbiosis. In total, nine filamentous fungi and one yeast were identified using morphological characteristics and DNA sequence data. Neocosmospora metavorans (Hypocreales: Nectriaceae), Fusarium sp. (Hypocreales: Nectriaceae), that is undescribed, and Meyerozyma guilliermondii (Saccharomycetes: Saccharomycetales) (yeast) were isolated more frequently from the head (including from mycangia, the fungus-carrying organ) than from the thorax and abdomen of adult beetles. Neocosmospora metavorans was the dominant species isolated from 12 out of 16 heads at 200 to 3300 CFUs/head, compared to the primary mycangia fungus from wild beetles, i.e., Fusarium kuroshium (Hypocreales: Nectriaceae). Temperature had a marked effect on fungal growth in the three symbiont species. Our results represent a major paradigm shift in understanding beetle–fungal interactions, as they show specific symbiont switching can occur in different nesting places.

1. Introduction

Symbiotic association between insects and microbes is critically important for the reproductive success of newly founded insect colonies [1,2]. Ambrosia beetles have several symbionts, including filamentous fungi and yeasts, bacteria, and viruses [3]. Symbiotic ambrosia fungi are often carried by mycangia (singular form: mycangium) of the female ambrosia beetle [4], and are introduced into host trees during gallery construction [5]. Ambrosia beetles obtain essential nutrients from symbionts in the low nutrient environment within the sapwood of their host tree [6]. Thus, the symbionts of ambrosia beetles represent a rich source of nutrients [7,8,9,10].
Ambrosia beetles are not particularly suitable for study because of their cryptic lifestyle and the uncontrolled environment in wild conditions [11]. Compared to wild populations, laboratory-reared ambrosia beetles can be used for several purposes in studies related to ecology, ethology, physiology, and biological control [12,13,14,15,16]. Rearing systems are human-made ecosystems that can be controlled for food and environmental factors, including temperature, humidity, lighting, gas exchange, water, and the type of containers used [17,18] to create homogenous and consistent conditions for development [14,19]. Several studies have shown that in addition to factors such as pH, host specificity, and life stage, the rearing substrate also determines the composition of the microbial communities in the gut of ambrosia beetle [18]. Experimental evidence suggests that altering the diet of ambrosia beetles influences symbiont community structure [14,20]. Furthermore, the ambrosia beetle’s microbiota can influence its essential traits, nutrition, and reproduction [12,21]. The microbiota has proved to be highly relevant for some invasive ambrosia beetle species, and has a wide host range but few reproductive host types [18,22].
The ambrosia beetle, Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae), is a wood-boring pest found in tree species such as poplar trees in Argentina and China, and box elder trees (Acer negundo L.) in the United States [23,24,25,26]. In Japan, E. interjectus is a vector of Ceratocystis ficicola Kajitani et Masuya (Microascales: Ceratocystidaceae) [27,28,29], a pathogenic fungus that causes wilt disease in fig trees (Ficus carica L.) [28,30,31,32]. Recently, we reported that the mycangia of E. interjectus were located in the oral cavity [33], and Fusarium kuroshium (Na, Carrillo et Eskalen ex Sand.-Den. et Crous) (Hypocreales: Nectriaceae) was closely associated with the wild adult female of E. interjectus [34]. However, the symbiont of E. interjectus grown in a rearing system established using the method of Mizuno and Kajimura [17] has not been evaluated.
We aimed to evaluate the fungal community in mycangia of reared E. interjectus compared to its wild population [34] to obtain a better understanding of beetle–fungus symbiosis and obtain useful information on the wilt disease of host trees. Towards this end, fungal species were isolated and identified in the head, thorax, and abdomen of female adults that were administered an artificial diet. Further, the phylogeny, dominance, and abundance of the isolated fungi were analyzed quantitatively.

2. Materials and Methods

2.1. Ambrosia Beetle Collection

Adult female E. interjectus specimens to be used for fungal isolation were reared on semi-artificial diets with a two-layer structure as described by Mizuno and Kajimura [17] at 25 °C in dark conditions to obtain successive generations of the beetle at the Forest Protection Laboratory of Nagoya University (Figure 1). The original mother beetle was collected from a fig tree with C. ficicola in a fig orchard in Hiroshima Prefecture, western Japan, in 2009. A total of 54 individuals from the reared population were collected between 11 September 2017 and 3 November 2017, of which 38 individuals were used for isolating fungal symbionts and 16 individuals (including 3 for the preliminary test) were used for culturing the symbionts for quantitative analysis.

2.2. Fungal Isolation and Culturing

Potato dextrose agar (PDA: 4 g potato starch, 20 g dextrose, 15 g agar, sterile distilled water up to 1 L; Difco) supplemented with streptomycin sulfate (0.1 g) was used after autoclaving at 121 °C for 15 min. Sterile Petri dishes (INA-OPTIKA Co., Ltd., Osaka, Japan) were prepared using 10 mL of PDA solution and kept in a sterile laminar flow chamber under UV light until the culture medium solidified.
Live specimens were stored at 15 °C with water-moistened sterile filter paper for 2 days after collection. The surfaces of whole beetles were washed by vortexing for 15 s in 1 mL sterile distilled water and one small drop of Tween-20 (<10 μL) [35]. A second wash was performed using sterile distilled water alone. After washing, the beetles were placed on sterile filter paper to remove water droplets from the body surface, and then were dissected. Each washed beetle was separated to obtain its head, thorax, and abdomen using two sterile pins under an Olympus SZ6045-TRPT stereomicroscope (Olympus Optical Co., Ltd., Tokyo, Japan). Next, the three body parts were inoculated on PDA plates.
Isolates were incubated at 25 °C, in dark conditions. After 5–7 days, the growth was checked, being careful not to miss even tiny colonies. Each colony was picked with a sterile toothpick and inoculated onto three PDA plates. If morphologically different colonies appeared among these three plates, the same process was repeated; purification was complete when identical colonies could be clearly visible on all three plates. One of the purified colonies was selected and allowed to grow sufficiently under the culture conditions described above for sub-identification. The total number of sub-identified colonies, the dominant morphotypes of which were stored on PDA slants at 25 °C, was recorded for each body part isolated.

2.3. Fungal Identification

The isolates obtained were initially characterized based on the morphology of the fungal colony and were grouped based on similarities in the morphotype (e.g., growth speed, color, thickness, transparency, and texture). The isolates were examined and photographed using the phase-contrast Olympus BX41 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) and an Olympus FX380 3CCD digital camera system with the FLvFs software (Flovel Image Filling System version 2.30.03, Tokyo, Japan). Fungal identification was performed based on morphological characteristics (e.g., conidial spores). The data was further supported via sequencing of the internal transcribed spacer (ITS) rDNA. For ambrosia Fusarium, the sequence data of 3 genes, that is, the translational elongation factor 1-α (TEF1), DNA-directed RNA polymerase II largest subunit (RPB1), and the second-largest subunit (RPB2) were evaluated along with the ITS.

2.4. DNA Extracting and Sequencing

The mycelium of each isolate was harvested from two-week-old plates and DNA was extracted using the PrepMan® Ultra Sample preparation reagent (Applied BiosystemsTM, Waltham, MA, USA) as per the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification was performed using the extracted DNA with the primers ITS5/ITS4 [36] for ITS rDNA, EF1/EF2 [37] for TEF1, newly designed primers, AF-RPB1F (5′-TTCCTCACCAAGGAGCAGAT-3′)/AF-RPB1R (5′-TCGCCAATAACATGGTCAAA-3′) for RPB1, and AF-RPB2F (5′-ACGATCCATGGAGTTCCTCA-3′)/AF-RPB2R (5′-CGTTGTACATGACCTCGAAA-3′) for RPB2. Twenty µL of the PCR mix consisted of 10 µL of GoTaq master mix (Promega Co., Ltd.), 1 µLof DNA template, 0.5 µL of each primer (10 mM), and 8 µL of sterile distilled water. The PCR conditions included an initial denaturation at 95 °C for 4 min, 40 cycles of 30 s at 94 °C, 30 s at 53 °C (annealing temperature), and 50 s at 72 °C, with a final elongation of 72 °C for 8 min for the ITS regions. For other sequence regions, appropriate annealing temperatures were used including 55 °C for EF1/EF2 and AF-RPB2, and 48 °C for AF-RPB1. Amplicons were confirmed by running the PCR product on a 1% agarose gel with GelRedTM Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA). The PCR products were purified using the ExoSAP-IT PCR Product Cleanup reagent (Applied BiosystemsTM) as per the manufacturer’s instructions, and the DNA obtained was sequenced from both ends using the BigDye Terminator v. 3.1 ready reaction mixture (Perkin-Elmer, Warrington, UK). Sequence data were obtained using the ABI PRISMTM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences were assembled using CAP3 [38], combined in each species, edited on the Aliview [39], and used for phylogenetic analysis.

2.5. Phylogenetic Analyses

For the in-depth identification of ambrosia Fusarium, phylogenetic analysis was performed. Sequence datasets for use in the alignment were produced using the obtained sequences and that of related species present in the NCBI database (accession numbers are included in Supplementary Table S1). The sequences were aligned using the MUSCLE algorithm [40] and adjusted manually. The maximum-likelihood tree prepared using an ultrafast bootstrap of 1000 replicates [41] was inferred using the IQ-TREE program [42] with the Model Finder option [43].

2.6. Quantitative Culturing of Fungi

Based on a previous examination of the oral mycangia of E. interjectus [33,44], the head of the beetle was targeted for quantitative culturing of fungi from its mycangia. The head was separated using two sterile pins as described above and transferred into 1.5 mL microcentrifuge tubes containing 0.5 mL of sterile distilled water and crushed using a sterilized micro-pestle.
A 0.1 mL of water from the homogenized head was serially diluted (1/10, 1/100, and 1/1000 fold dilutions), and 0.1 mL of the water from each dilution was plated onto PDA. Each treatment was repeated 7 times. Fungal colonies on the PDA plates were grown in an incubator at 25 °C, in dark conditions to enable observation of fungal colony characteristics. The plates were monitored every 2–3 days during the two-week incubation and marked with different color pens to ensure the consistent assignment of morphotypes based on the micro-morphology (i.e., color, size, growth rate, and texture) of the colony. The marking was used for the dilutions 1/10 and 1/100 on PDA. Fungal species were confirmed via phylogenetic analyses as described above. Colony-forming units (CFUs) of each PDA were recorded, and the mean was computed using the CFU data excluding the maximum and minimum values (n = 5).

2.7. Fungal Growth

In this study, the optimal growth temperature of the tested fungi was considered as that required to reach maximum size at the fastest rate when grown on the Petri dish which contained artificial media. To identify the optimal growth temperature, three species of the dominant fungal morphotype (N. metavorans, Fusarium sp., and M. guilliermondii) isolated from the head of E. interjectus were tested. Each selected fungus was inoculated on a 9 cm Petri dish containing PDA and grown at 25 °C in the dark for 5 days to enable the development of primary cultures. The fungal colony discs (Ø 0.5 cm; N. metavorans and Fusarium sp.) from primary cultures were transferred and placed in the center of the prepared PDA Petri dish. Using a sterile toothpick, yeast was inoculated (Ø 0.05 cm; M. guilliermondii) from primary cultures onto the prepared PDA and grown until saturation. Seven PDA Petri dishes, each corresponding to one fungus matrix, were prepared for the tested fungi. The dishes were incubated at seven different temperatures (i.e., 5, 10, 15, 20, 25, 30, and 35 °C; dark conditions) between 20 February 2018 and 20 March 2018.
Two perpendicular straight lines were drawn on the underside of each Petri dish. The crossing point coincided with the center of the 0.5 cm initial fungal disc. Radial growth measurements were recorded every second day from the edge of the initial inoculum to the periphery of mycelial development along the four segments formed by the two perpendicular lines. Data for every second day corresponded to the mean of four measurements. Bioassays were ended when the mycelia reached the wall of the Petri dish in any dish.

2.8. Subsection

Statistical analyses were performed using the SPSS version 19.0 software (IBM Corporation, Armonk, NY, USA, 2010). The Kruskal–Wallis test was used to evaluate the differences in the surface area (cm2) of the fungal colony on the 6th day after inoculation across the seven temperature treatments for each tested fungus.
Visible density (VD, CFUs/head) of associated fungi isolated from the head, relative dominance (RD, %), and frequency of occurrence (FO, %) of fungal species isolated from the head, thorax, and abdomen of adult E. interjectus females were calculated using the equations listed below:
VD   ( CFUs / head ) = Number   of   CFUs   plated   on   PDA   ×   Dilution   factor   ×   Original   sample   factor     Volume   of   culture   plated   on   PDA   ,
where the dilution factor is 10 or 100, original sample factor is 0.5 mL including 1 head of the adult female, and the volume of culture plated on PDA is 0.1 mL.
RD   ( % ) = Number   of   fungal   isolates   of   each   species Total   number   of   fungal   isolates   of   all   the   species × 100
FO   ( % ) = Number   of   beetles   from   which   each   fungal   species   was   isolated Total   number   of   the   beetles   used   for   isolation × 100

3. Results

3.1. Fungal Flora

After morphological evaluation, 197 fungal isolates were obtained from 54 adult E. interjectus females. The source of isolation for the fungi included 69, 62, and 66 isolates from the head, thorax, and abdomen, respectively, which were sequenced. Ten species, including nine filamentous fungi, were identified: Neocosmospora metavorans (Al-Hatmi et al.) (Hypocreales: Nectriaceae), Fusarium sp., Byssochlamys nivea Westling (Eurotiales: Trichocomaceae), Penicillium citrinum Thom (Eurotiales: Trichocomaceae), Phaeoacremonium inflatipes Gams, Crous et Wingfield (Calosphaeriales: Calosphaeriaceae), Paecilomyces sinensis Chen, Xiao et Shi (Eurotiales: Trichocomaceae), Fusarium tricinctum (Corda) (Hypocreales: Nectriaceae), Arthrinium sp. (Sordariomycetes: Apiosporaceae) and Pestalotiopsis mangiferae (Henn.) (Xylariales: Amphisphaeriaceae), and the yeast Meyerozyma guilliermondii (Wick.) (Saccharomycetes: Saccharomycetales). The sequences of four isolates failed to amplify via PCR, and these were treated as unknown species (Figure 2 and Table 1).

3.2. Phylogenetic Analyses

We performed sequence alignments using the four sequences for ITS rDNA, TEF, RPB1, and RPB2 for phylogenetic analysis. The resulting alignment had 70 sequences with 2871 columns, 177 distinct patterns, 120 parsimony-informative, 51 singleton sites, and 2700 constant sites. We selected individual models for the four regions using BIC, including TIMe+R2 for ITS, TIM3e+I for TEF, Tne+G4 for RPB1, and K2P+I for RPB2. The best log-likelihood score was −5608.252. The phylogenetic tree showed that the Fusarium species isolated from the head including the oral mycangia of reared E. interjectus was placed in an independent clade within the ambrosia fusaria, and the classification was supported by high bootstrap values (Figure 3). Thus, phylogenetic analysis indicated that the Fusarium isolate obtained from the head including the oral mycangia of the reared adults of E. interjectus was an undescribed Fusarium species.

3.3. Relative Dominance and Frequency of Occurrence

The Relative dominance (RD) ranged from 1.4% to 46.4% (Table 1). The majority of isolates from the head included N. metavorans with an RD of 46.4%. The RD of N. metavorans from the thorax and abdomen was 35.5%. The frequency of occurrence (FO) of N. metavorans isolated from the head (59.3%) was higher than that of other fungi (1.9–24.1%). Neocosmospora metavorans was the most common species in adult E. interjectus females in the other body parts; the FO of N. metavorans from the thorax and abdomen was 40.7% and 42.6%, respectively.
The second and third most dominant species in the head were Fusarium sp. and M. guilliermondii, with an RD of 18.8% and 17.4%, respectively. The FO of Fusarium sp. and M. guilliermondii from the head was 24.1% and 22.2%, respectively. However, the RD and FO were lower in the thorax and abdomen relative to the head, and B. nivea was the second-dominant species.

3.4. Quantitative Culturing

The 16 beetle (A-P) macerated heads, which were crushed using sterile water, yielded four fungal morphotypes on PDA. The four species could be distinguished by color, mycelial texture, and relative growth rate, as described previously (Figure 2 and Table 1), and included N. metavorans (12 beetles), M. guilliermondii (10 beetles), Fusarium sp. (1 beetle), and B. nivea (1 beetle) (Table 2).
Neocosmospora metavorans was isolated from 12 out of 16 heads with a mean of 360 to 2620 CFUs/head (Table 2). Meyerozyma guilliermondii was recovered from 10 out of 16 heads at a mean of 115 to 27,500 CFUs/head. Fusarium sp. and B. nivea were isolated from only 2 individuals of L and D (920 and 1490 CFUs/head, respectively).

3.5. Fungal Growth

The results are summarized in Figure 4. In the N. metavorans, Fusarium sp., and M. guilliermondii, the median surface area of the fungal colony was significantly different across the various temperatures analyzed (Kruskal–Wallis test, p < 0.001). The optimal growth temperature was 25 °C for Fusarium sp. and 30 °C for N. metavorans, and these fungal species were unable to grow at 5 °C. In contrast, M. guilliermondii formed very small colonies (Ø 0.4–1.23 cm) at all temperatures, with optimal growth at 35 °C (Figure 4).
Colony macro-morphology analysis on PDA showed that N. metavorans colony was white, had irregular edges, and reached the edge of the cellophane membrane 10 days after inoculation at 25, 30, and 35 °C. Fusarium sp. colony was white overall but with hazel coloring in the middle area and regular edges, and reached the edge 12 days after inoculation at 20 and 25 °C. Meyerozyma guilliermondii colony was milky white at 5–25 °C, but was light yellow at 30 and 35 °C (Figure 2 and Figure 4, and Supplementary Figure S1).

4. Discussion

In this study, we report for the first time the fungal flora associated with reared E. interjectus. In a previous study [45], the characteristics of the symbiotic fungi of ambrosia beetles, in terms of contribution as food sources, were classified into two broad categories; i.e., primary ambrosia fungi (PAF) and auxiliary ambrosia fungi (AAF). Neocosmospora metavorans, Fusarium sp., and M. guilliermondii isolated from the head were more frequent than in the thorax and abdomen of the examined beetles (Table 1). Considering that E. interjectus has oral mycangia in its head [33], the beetle will act as a carrier for the abovementioned dominant fungi which may serve as the PAF of E. interjectus in artificial diets. Other fungi, including Arthrinium sp., Byssochlamys nivea, Fusarium tricinctum, Paecilomyces sinensis, Penicillium citrinum, Pestalotiopsis mangiferae, and Phaeoacremonium inflatipes were isolated with low frequency, and classified as AAF; therefore, these were not considered further as candidates.
In the wild population, which was collected from fig trees in the Hiroshima prefecture, F. kuroshium was closely associated with the adult E. interjectus female and was the most dominant in the head, including the oral mycangia [34]. In the reared population, Fusarium sp., which is distinct from F. kuroshium, was isolated from the adults. Additionally, N. metavorans was more dominant than Fusarium sp. These results suggest that an artificial diet is more suitable for N. metavorans than the fig tree, which may eventually result in changes to the fungal flora. Thus, different nesting places from the fig tree to an artificial diet may change the dominance of fungal species in E. interjectus. In the wild and reared populations, the number of fungal species isolated from the thorax and abdomen was more than that from the head (Table 1 and Figure 5). The surface structure of the abdomen (elytra) may accidentally trap fungal spores, which can then be transported to the fig trees.
Our results show that the beetle’s mutualists (N. metavorans, Fusarium sp., and M. guilliermondii) were sensitive to temperature (Figure 4). Thus, we confirm and further support the findings that temperature adaptation (e.g., climate) plays a crucial role in the population dynamics of the ambrosia beetle–fungal symbionts [46,47,48]. Furthermore, the temperature preference indicates that these fungi (as well as their hosts) are adapted to colonize thicker or warmer trees [49,50]. This may explain why E. interjectus–Fusarium sp. prefers to infest old (thick) fig trees (>20 years old) rather than young (thin) fig saplings in fig orchards in Japan [30]. Our findings can be used to predict the adaptive capabilities of fungal symbionts in various growing conditions [51]. Additional information relating to the internal temperatures of preferred host trees of E. interjectus, such as fig, poplar, and box elder trees can be obtained in the future to evaluate the actual growth conditions of these fungi.
Previous studies have shown that F. kuroshium and N. metavorans (formerly known as the Fusarium solani species complex (FSSC) phylogenetic species 6) showed a symbiotic association with ambrosia beetles [52,53,54,55,56,57,58]. Fusarium kuroshium associated with Euwallacea sp. forms a monophyletic group (ambrosia Fusarium clade) [54,59,60] and caused wilt and dieback of avocado in diverse landscape trees in concert with a mass attack by Euwallacea sp. in Israel and California, USA [61,62]. FSSC was reportedly associated with other Scolytidae, including Euwallacea fornicatus (Eichhoff) [53], Xyleborus ferrugineus (Fabricius) [52], and Xylosandrus compactus (Eichhoff) [55], and had a wide plant host range, including avocado (Persea americana Miller) [53], coffee (Coffea arabica L.) [63], Japanese cheesewood (Pittosporum tobira (Thunb. ex Murray)) [64], and the Indian coral tree (Erythrina variegata L.) [65,66]. Euwallacea interjectus shows an exclusive relationship with Fusarium floridanum Aoki, Smith, Kasson, Freeman, Geiser et O’Donnell (Hypocreales: Nectriaceae) on box elder in Florida, USA [25] and F. kuroshium on fig trees in Hiroshima, Japan [34]. Notably, in this study, N. metavorans in rearing conditions was classified as a unique fungal symbiont of E. interjectus. Therefore, E. interjectus can switch its fungal symbiont when reared on an artificial diet. The “host switch” phenomenon in F. kuroshium and N. metavorans under complex conditions requires further analysis to identify the effects on host adaptation.
The yeast M. guilliermondii, which was isolated from Platypus koryoensis (Murayama) (Coleoptera: Curculionidae), was reported from Korea [67]. Compared with Fusarium sp. and N. metavorans, M. guilliermondii may play an essential role in the ecology of ambrosia beetles. Potentially, the yeast associates of E. interjectus may differentially influence the ability of the beetle to respond to changing environmental conditions, population dynamics, and outbreak behavior [68].
The oral mycangia of E. interjectus are roughly spherical, approximately 100 μm in diameter, and tightly packed with compacted fungal inoculum [33,44]. Although gland cells around the mycangia were not identified, similar mycangia in other ambrosia beetles are filled with glandular secretions that may foster growth and budding of fungal spores [2,10,54,69,70,71]. According to a previous report, assuming the size of the smallest spore of M. guilliermondii is approximately 2 μm in diameter [72,73], we estimate that approximately 250,000 spores of M. guilliermondii can be packed into the paired oral mycangia in the head. The estimates of spore numbers for M. guilliermondii in E. interjectus are conservative because sample grinding was not complete, and a few spores may adhere to beetle tissue or the glass walls of grinders, and a few spores may likely be killed during the storage of the beetles or grinding process. Nonetheless, thousands of viable spores of M. guilliermondii were recovered from individual, surface-sterilized heads of E. interjectus. The high number of M. guilliermondii CFU (≤43,000 CFUs/head) can be obtained if the mycangia are tightly packed and collected in the budding yeast phase (Table 2). Sporulation within mycangia may serve as an important adaptation for the fungal symbionts [67], and competition among the M. guilliermondii may be keen. Future studies should focus on the functions of yeast in E. interjectus development and fungal interactions, especially for yeast diversity. A better understanding of the complex symbiotic relationship of ambrosia beetles with microorganisms will further aid artificial control efforts in the rearing system.
To conclude, we evaluated fungal flora in adult females of the reared ambrosia beetle E. interjectus. This study is the first report of fungal symbiont diversity associated with the reared ambrosia beetle E. interjectus. Further, we identified N. metavorans as a dominant fungal species in the head (including oral mycangia) of the reared adult female of E. interjectus. These results represent a major paradigm shift in our understanding of beetle–fungal interactions and show that specific symbiont switching can occur in different nesting places.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d14070535/s1, Figure S1: Representative images of three studied fungi (A: Neocosmospora metavorans, B: Fusarium sp., and C: Meyerozyma guilliermondii) growing on PDA treated with four investigated temperature treatments (15, 20, 25, and 30 °C) on the 6th and 10th day after inoculation. The three remaining treatments (5, 10, and 35 °C) did not work for fungal colony formation as shown in Figure 4; Table S1: Accession numbers of sequences for ambrosia Fusarium used for phylogenetic analysis.

Author Contributions

H.K. and Z.-R.J. conceived the study. H.K. reared and collected the beetle populations. Z.-R.J. isolated the fungi, analyzed the data, and wrote the early version of the manuscript. H.M. sequenced the fungi and constructed the phylogenetic trees. H.K. supervised the study and reviewed the manuscript. All authors contributed to the study. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by JSPS (the Japan Society for the Promotion of Science) KAKENHI Grant (Grants-in-Aid for Scientific Research) Numbers 18KK0180, 19H02994, and 20H03026.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Acknowledgments

We thank Takahiko Mizuno for his help with the preparation of the semi-artificial diets, Shodai Jikumaru and Takeshige Morita for collecting the original mother beetle, Naoki Hijii and Wataru Toki for their assistance with result analysis, and members of the Forest Protection Laboratory at Nagoya University for their valuable suggestions regarding this work.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Typical reproduction of an ambrosia beetle, Euwallacea interjectus, on a semi-artificial diet. (A) Progeny (egg, larva, pupa, and new adult) in a gallery that the dispersed female adult (their mother) oviposited after the establishment of ambrosia fungi in the diet. (B) Female adult of E. interjectus in lateral views which emerged on the diet. (C) Micro-CT scanning cross-section of oral mycangium (colored in green) in the female adult by Jiang et al. [33] (unpublished image): in its dispersal period, the mycangium has filled with compacted fungal inoculum (colored in red), which is used to propagate a fungal garden in newly-bored galleries. Scale = 1 mm.
Figure 1. Typical reproduction of an ambrosia beetle, Euwallacea interjectus, on a semi-artificial diet. (A) Progeny (egg, larva, pupa, and new adult) in a gallery that the dispersed female adult (their mother) oviposited after the establishment of ambrosia fungi in the diet. (B) Female adult of E. interjectus in lateral views which emerged on the diet. (C) Micro-CT scanning cross-section of oral mycangium (colored in green) in the female adult by Jiang et al. [33] (unpublished image): in its dispersal period, the mycangium has filled with compacted fungal inoculum (colored in red), which is used to propagate a fungal garden in newly-bored galleries. Scale = 1 mm.
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Figure 2. Morphological characteristics of Fusarium sp., Neocosmospora metavorans, and Meyerozyma guilliermondii. (A,B): colony and conidial spores of Fusarium sp., respectively, on PDA (25 °C, dark, 12 days); (C,D): colony and conidial spores of N. metavorans, respectively, on PDA (25 °C, dark, 14 days); (E,F): colony and conidial spores of M. guilliermondii, respectively, on PDA (25 °C, dark, 9 days).
Figure 2. Morphological characteristics of Fusarium sp., Neocosmospora metavorans, and Meyerozyma guilliermondii. (A,B): colony and conidial spores of Fusarium sp., respectively, on PDA (25 °C, dark, 12 days); (C,D): colony and conidial spores of N. metavorans, respectively, on PDA (25 °C, dark, 14 days); (E,F): colony and conidial spores of M. guilliermondii, respectively, on PDA (25 °C, dark, 9 days).
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Figure 3. Multilocus phylogenetic analysis of ambrosia Fusarium clade conducted with four genes: ribosomal internal transcribed spacer, elongation factor 1-a, DNA-directed RNA polymerase II largest subunit, and DNA-directed RNA polymerase II second largest subunit. The diagram was constructed using IQ-TREE maximum likelihood method bootstrapped with 1000 replications. The red box indicates the phylogenetic placement of the Fusarium species isolated from the head including oral mycangia of the reared adults of E. interjectus.
Figure 3. Multilocus phylogenetic analysis of ambrosia Fusarium clade conducted with four genes: ribosomal internal transcribed spacer, elongation factor 1-a, DNA-directed RNA polymerase II largest subunit, and DNA-directed RNA polymerase II second largest subunit. The diagram was constructed using IQ-TREE maximum likelihood method bootstrapped with 1000 replications. The red box indicates the phylogenetic placement of the Fusarium species isolated from the head including oral mycangia of the reared adults of E. interjectus.
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Figure 4. Growth of mycelial cultures of (A) Neocosmospora metavorans, (B) Fusarium sp., and (C) Meyerozyma guilliermondii on the PDA at different temperature treatments (5, 10, 15, 20, 25, 30, and 35 °C). (A-1,B-1,C-1) are the fungal colony’s surface area of N. metavorans, Fusarium sp., and M. guilliermondii on the 6th day after inoculation in each temperature treatment, respectively. The overall difference in medians among the temperature treatments (the 6th day after inoculation) is significant at p < 0.001 using Kruskal–Wallis test. Means with different letters (a, b, c, d, e, and f) are significantly different among temperatures at the 1% level. SD, standard deviation. N = 5 replicates per fungal species.
Figure 4. Growth of mycelial cultures of (A) Neocosmospora metavorans, (B) Fusarium sp., and (C) Meyerozyma guilliermondii on the PDA at different temperature treatments (5, 10, 15, 20, 25, 30, and 35 °C). (A-1,B-1,C-1) are the fungal colony’s surface area of N. metavorans, Fusarium sp., and M. guilliermondii on the 6th day after inoculation in each temperature treatment, respectively. The overall difference in medians among the temperature treatments (the 6th day after inoculation) is significant at p < 0.001 using Kruskal–Wallis test. Means with different letters (a, b, c, d, e, and f) are significantly different among temperatures at the 1% level. SD, standard deviation. N = 5 replicates per fungal species.
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Figure 5. Comparison of fungal flora and their relative dominance in female adults of Euwallacea interjectus among wild ((a): 51 beetles) [34] and reared ((b): 54 beetles) populations (Table 1). Numbers of beetles from which each fungal species was isolated is shown in pie charts.
Figure 5. Comparison of fungal flora and their relative dominance in female adults of Euwallacea interjectus among wild ((a): 51 beetles) [34] and reared ((b): 54 beetles) populations (Table 1). Numbers of beetles from which each fungal species was isolated is shown in pie charts.
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Table
Table 1. Relative dominance (RD, %) and frequency of occurrence (FO, %) of fungal species isolated from head, thorax, and abdomen of reared female adults of Euwallacea interjectus in this study.
Table 1. Relative dominance (RD, %) and frequency of occurrence (FO, %) of fungal species isolated from head, thorax, and abdomen of reared female adults of Euwallacea interjectus in this study.
Fungal SpeciesNo. of Fungal Isolates and Relative Dominance (RD, %) a in Each Body Part No. of Beetles from Which Each Fungal Species Was Isolated and Frequency of Occurrence (FO, %) b in Each Body Part
HeadThoraxAbdomen HeadThoraxAbdomen
Neocosmospora metavorans32(46.4)22(35.5)23(34.8) 32(59.3)22(40.7)23(42.6)
Fusarium sp.13(18.8)2(3.2)5(7.6) 13(24.1)2(3.7)5(9.3)
Meyerozyma guilliermondii12(17.4)3(4.8)1(1.5) 12(22.2)3(5.6)1(1.9)
Byssochlamys nivea5(7.2)18(29.0)17(25.8) 5(9.3)18(33.3)17(31.5)
Penicillium citrinum1(1.4)4(6.5)10(15.2) 1(1.9)4(7.4)10(18.5)
Paecilomyces sinensis—1(1.6)4(6.1) —1(1.9)4(7.4)
Phaeoacremonium inflatipes—1(1.6)— —1(1.9)—
Pestalotiopsis mangiferae——1(1.5) ——1(1.9)
Arthrinium sp.—1(1.6)— —1(1.9)—
Fusarium tricinctum—1(1.6)— —1(1.9)—
Unknown 32(2.9)2(3.2)— 2(3.7)2(3.7)—
Unknown 4—1(1.6)1(1.5) —1(1.9)1(1.9)
Unknown 54(5.8)6(9.7)3(4.5) 4(7.4)6(11.1)3(5.6)
Unknown 6——1(1.5) ——1(1.9)
Total c696266Number d545454
a RD (%) = Number   of   fungal   isolates   of   each   species Total   number   of   fungal   isolates   of   all   the   species × 100 % . b FO (%) = Number of beetles from which each fungal species was isolated/Total number of the beetles used for isolation × 100%. c Total number of fungal isolates of all the species. d Total number of beetles tested. —not detected.
Table
Table 2. Visible density (VD, CFUs/head) of associated fungi isolated from the head of reared female adults Euwallacea interjectus.
Table 2. Visible density (VD, CFUs/head) of associated fungi isolated from the head of reared female adults Euwallacea interjectus.
Beetle IndividualVisible Density (VD, CFUs/Head) a
Neocosmospora metavoransMeyerozyma guilliermondiiFusarium sp.Byssochlamys nivea
MeanMinMaxMeanMinMaxMeanMinMaxMeanMinMax
A———18,000950023,500——————
B1290900200027050400——————
C36020060011550200——————
D———19050900———149010001850
E6503501000200100250——————
F15404003050830501500——————
G202015002300—————————
H224016503150—————————
I262018503300—————————
J———27,50022,50043,000——————
K12908001500—————————
L———140502509205501400———
M1700125023501703004150——————
N179010002250—————————
O155012001800—————————
P7505001250212018002350——————
a Visible density (VD, CFUs/head) was calculated as follows: (NC×DF×OS)/VC, NC equals the number of colonies forming units (CFUs) plated on PDA; DF equals dilution factor of 10 or 100, OS equals the original sample factor, i.e., 0.5 mL volume of sample is equivalent to 1 head of ambrosia beetle; VC equals the volume of culture plated on PDA, i.e., 0.1 mL in this study. — not detected
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Jiang, Z.-R.; Masuya, H.; Kajimura, H. Fungal Flora in Adult Females of the Rearing Population of Ambrosia Beetle Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae): Does It Differ from the Wild Population? Diversity 2022, 14, 535. https://doi.org/10.3390/d14070535

AMA Style

Jiang Z-R, Masuya H, Kajimura H. Fungal Flora in Adult Females of the Rearing Population of Ambrosia Beetle Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae): Does It Differ from the Wild Population? Diversity. 2022; 14(7):535. https://doi.org/10.3390/d14070535

Chicago/Turabian Style

Jiang, Zi-Ru, Hayato Masuya, and Hisashi Kajimura. 2022. "Fungal Flora in Adult Females of the Rearing Population of Ambrosia Beetle Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae): Does It Differ from the Wild Population?" Diversity 14, no. 7: 535. https://doi.org/10.3390/d14070535

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