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Effects of different edible mushroom hosts on the development, reproduction and bacterial community of Tyrophagus putrescentiae (Schrank)
Journal of Stored Products Research 61 (2015) 70e75
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Effects of different edible mushroom hosts on the development, reproduction and bacterial community of Tyrophagus putrescentiae (Schrank) S.-X. Qu a, b, H.-P. Li b, L. Ma b, L.-J. Hou b, J.-S. Lin b, J.-D. Song b, X.-Y. Hong a, * a b
Department of Entomology, Nanjing Agricultural University, Nanjing 210095, China Institute of Vegetable Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 March 2014 Received in revised form 27 November 2014 Accepted 12 December 2014 Available online 19 December 2014
The storage mite, Tyrophagus putrescentiae (Schrank), infests various stored products, plant seeds, chinese herbal medicines and edible mushrooms in China. To assess interactions between edible mushroom hosts and the mite, we investigated the effects of nine species on development and reproduction at 22 Cand 85% humidity in the laboratory. We also demonstrated changes in the bacterial community of T. putrescentiae reared on four mushroom hosts. All the immature stages, female longevity and reproductive periods were significantly affected by the mushroom species, total immature developmental time varied from 4.41 ± 0.20 (reared on Auricularia polytricha) to 9.79 ± 0.92 days (when reared on Ganoderma lucidum). The egg stage was the longest period of the immature stages, whereas the protonymph and tritonymph stages were the shortest at about 2 days. The net reproductive rate (R0), and the intrinsic rate of increase (rm) were significantly different on the nine mushroom hosts. Both the maximum fecundity (212.85 eggs per female) and the highest rm were obtained on A. polytricha. The results indicated that T. putrescentiae had a wide range of adaptability to edible mushrooms, with development and reproduction of the mite being significantly affected by different species. Changes in the bacterial communities inhabiting the mites were influenced by mushroom hosts. A total of 30 full length sequences of 16s rRNA gene were obtained from the mite reared on four hosts. The bacterial community inhabiting T. putrescentiae on A. polytricha host was the most diverse with six different species, two of these genera occurred on Flammulina velutipes host. Consequently the interactions between T. putrescentiae and mushroom hosts can influence both the biological characteristics and bacterial community. The changes of bacterial community might participate in the different fungal mycelium hydrolysis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Mushroom pest Trophic levels Storage mite Flour mite
1. Introduction Tyrophagus putrescentiae (Schrank) belongs to the class Arachnida (Acarina, acaridida, Acaridae, Tyrophagus). It is one of the most common synanthropic mites with a cosmopolitan occurrence, and is a major pest of stored grain. It attacks a very wide range of produce including rice, wheat bran, herbs, cheese, nuts and candied fruit, peanut, and flour, as well as edible fungi and mushroom beds (Zhang and Jin, 1960; Hughes, 1976; Zou, 1987; Eraky, 1995; Chmielewski, 1999). Its feeding on fungi has been widely reported
* Corresponding author. E-mail address: [email protected] (X.-Y. Hong). http://dx.doi.org/10.1016/j.jspr.2014.12.003 0022-474X/© 2014 Elsevier Ltd. All rights reserved.
and it can develop on many fungal species (Smrz and Catska, 1989; Zhang et al., 1992; Hubert et al., 2004). The mite can also promote fungal growth by removing senescent parts of the mycelium which stimulates fungal metabolism (Hatcher, 1995; Hubert et al., 2003). T. putrescentiae is able to transfer Aspergillus flavus (Link) to sterile grain, resulting in fungus growth and aflatoxin production (Franzolin et al., 1999). The latest study had shown that the mite transmited an amycotoxin-producing strain of Fusarium poae (Peck) Wollenw to stored barley, and increased the deoxynivalenol (DON) content in the barley following successful F. poae transfer (Hubert et al., 2014). With such a wide range of hosts and suitable habitats, T. putrescentiae is a major pest of mushroom cultivation and storage (Lu, 2002). According to statistics, its mushroom hosts include
S.-X. Qu et al. / Journal of Stored Products Research 61 (2015) 70e75
Lentinula edodes (Berk), Agaricus bisporus (Lange), Pleurotus ostreatus (Kumm), Ganoderma sp., and Pleurotus pulmonarius (Fr.). It can carry and spread pathogens, reduce mushroom production, cause dried mushroom mildew, and cause human allergic dermatitis, pulmonary acariasis, and intestinal acariasis (Li et al., 2003; Jeong et al., 2007), seriously affecting the economic efficiency of mushroom production and the health of mushroom farmers. According to the Ministry of Agriculture, Chinese edible mushroom output has reached more than 10 million tons annually in recent years, accounting for over 70% of the total global production. As many as 50 varieties of edible mushroom are cultivated in China, 20 million people are engaged in the industry (Lu and Li, 2012). So far, only one study has reported the effects of temperature and edible fungi on development, life table, and thermal requirements of T. putrescentiae. The net reproductive rate (R0), and the intrinsic rate of increase (rm), were significantly different on the two mushroom hosts (Kheradmand et al., 2007). Bacteria can play beneficial, and often essential, roles in the lives of their hosts. In animals that carry vertically transmitted, intracellular bacteria, the host and its symbiont community form an inseparable holobiont with a shared metabolism and evolutionary fate (Wu et al., 2006; Moran, 2002). The aim of this study is to determine the effects of nine largest cultivated mushroom varieties on the development and reproduction of T. putrescentiae at 22 C and 85% humidity in the laboratory, and to demonstrate changes in the bacterial community inhabiting T. putrescentiae induced by different mushroom hosts.
2.1. Mites and mushroom culture The storage mites, T. putrescentiae used in the study were supplied from stock cultures originating from a population collected in 2012 on wood ear mushroom Auricularia polytricha (Mont.) in Feng County (Jiangsu, China), which had been kept in the laboratory at 25 C and approximately 85% RH. The mites used to assess host suitability were initially fed the nine edible mushrooms to be tested for four generations, to acclimatize them to each host. One week before the start of the experiments, the mushroom mycelia were inoculated onto potato dextrose agar (PDA) plates in 50 mm diameter Petri dishes. The dishes were covered with lids and placed in a growth chamber at
Table 1 List of edible mushroom species and strains from China used to investigate development, reproduction and bacterial communities in Tyrophagus putrescentiae. Species
Stain no.
Lentinula edodes Wuxiang No1 Agaricus bisporus AS2796 Pleurotus Za No 3 ostreatus P. eryngii Zhongnong var.tuoliensis No1 P. pulmonarius 206 Ganoderma lucidum Auricularia polytricha Flammulina velutipes Agrocybe cylindracea
Family and genus
Agaricales, Tricholomataceae, Lentinula Agaricales, Agaricaceae, Agaricus Agaricales, pleurotaceae, Pleurotus Agaricales, pleurotaceae, Pleurotus Agaricales, pleurotaceae, Pleurotus Taishan Aphyllophorales,Polyporaceae, Ganoderma Fengmao Auriculariales, Auriculariaceace, No 3 Auricularia Danbaijin Agaricales, Tricholomataceae, Flammulina Gucha No 1 Agaricales, Bolbitiaceae, Agrocybe
25 C and 60% RH in complete darkness. The mushroom species and strains used in the experiments are described in Table 1.
2.2. Development, reproduction and longevity Following inoculation of the nine mushroom mycelia onto the 50 mm PDA Petri dishes, a mating pair of mites was placed on each of the nine edible mushroom substrates. Ten replicates per mushroom species were used to determine the durations of the developmental stages of the mite lifecycle. Newly-laid (one-day old) eggs were separated with a hair brush onto five new 50 mm Petri dishes with the corresponding host mushroom mycelia using an S8APO dissection microscope (Leica, Germany). Each Petri dish received 5e7 eggs of the same age. Petri dishes were covered with Parafilm® (Bemis, USA) wrap and put into an SPX-2501C type artificial climate box (Suzhou Jiangdong Precision Instrument Co., Ltd.). All tests were performed in the laboratory at 22 ± 1 C, 85% ± 5% RH, in the dark. Observations of mite mortality and fecundity were made every day at 9:00 am and 15:00 pm.
2.3. Life-table parameters A life table was constructed from the observed survival and fecundity rates for individuals. Parameters were calculated by the methods of Birch (1948) and Roy et al. (2002). The intrinsic rate of increase (rm) was estimated by nonlinear regression according to:
X ½expð rm xÞlx mx ¼ 1
2. Materials and methods
Province of origin Zhejiang Fujiang Jiangsu
71
(1)
where rm is the intrinsic rate of increase, x is female age in days, lx is the fraction of females surviving to age x (age-specific survival rate), and mx is the expected number of daughters produced per female alive at age x (age-specific fecundity rate), obtained by multiplying the number of eggs by the age-specific sex ratio, which is defined as the proportion of females in the progeny. The net reproductive rate (R0), the mean generation time (t) in days, the finite rate of increase (l) and the doubling time (DT) were computed by the respective equations:
R0 ¼
X
lx mx
(2)
t ¼ ln R0 0=rm
(3)
l ¼ e rm
(4)
DT ¼ ln2=rm
(5)
One-way ANOVA was used to evaluate the effect of mushrooms hosts on developmental times, reproductive periods, female longevity and fecundity of T. putrescentiae. Means were compared by the SNK test at the 5% level using. These analyses were performed with SPSS 20.0.
Beijing Jiangsu
2.4. Bacterial communities inhabiting mites feeding on different edible mushroom species
Shandong Jiangsu Jiangsu Fujian
In this experiment, we used four different populations of the storage mite feeding on four mushroom species, A. bisporus, Pleurotus eryngii var. tuoliensis (Mou), A. polytricha, Flammulina velutipes (Fr.) to evaluate differences in their associated bacterial communities. Bacterial communities were characterized by 16S rRNA gene sequencing.
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2.4.1. DNA extraction, PCR amplification and sequencing Genomic DNA was extracted by homogenizing a single female adult using the Wizard®SV Genomic DNA Purification System (Promega, USA).The 16S rRNA gene was amplified using PCR, performed with the universal primers UF: 5-AGA GTT TGA TYM TGGC 3 (position 8e23) and UR: 5-GYT ACC TTG TTA CGA CTT (position 1496e1514) using a MyCycler™ Thermal Cycler (Bio-Rad, USA) (Barbieri et al., 2001). A total volume of 50 mL PCR reaction mixture contained 25 mL GoTaq® Green Master Mix (Promega, USA), 5 mL each of forward and reverse primers, and 3 mL of template DNA. Reactions were cycled 30 times at 94 C for 30 s, 50 C for 90 s, and 72 C for 1 min. The PCR products were electrophoresed on a 1.0% agarose gel in TBE for 20 min at 100 mA, and then purified with the AxyPrep™ PCR Clean-Up Kit (Axygen, USA). The PCR products were cloned using the pGEM-T easy vector system (Promega, USA) and transformed into Escherichia coli DH5a, and selected colonies from each plate were picked at random and sequenced by Sangon, Shanghai. 2.4.2. Phylogenetic analysis An initial alignment of all 16S rRNA gene sequences was performed using MEGA 5.1 (Hall, 2013). The Neighbor-Joining method was used based on the Jukes-Cantor distance matrix (1000 bootstrap resamplings) in MEGA 5.1, and the maximum-likelihood method with the Jones Taylor Thornton (JTT) model (1000 bootstrap resamplings). The resulting phylograms were finalized using MEGA 5.1 (Hall, 2011). 3. Results
other hosts needed at least 1e2 days to complete this stage. In Pleurotus, there were differences in the immature stages, except for the tritonymph stage. No differences were found in larvae, tritonymph and the total developmental time between P. eryngii var. tuoliensis and P. pulmonarius. 3.2. Longevity and reproductive period Females reared on different mushrooms were tested for the duration of their pre-ovipositional, ovipositional and postovipositional periods, and longevity (Table 3). The total longevity of females ranged from 35.1 ± 1.2 to 44.1 ± 2.5 days. Significant differences were observed among the nine hosts. The variation in post-ovipositional period reared on the nine different mushrooms was lustered into two groups without significant differences within them (F ¼ 9.620, p < 0.0001): among L. edodes, P. eryngii var. tuoliensis and P. pulmonarius; and among A. bisporus, P. ostreatus, G. lucidum, A. polytricha, F. velutipes, and A. cylindracea. Females had a shorter pre-ovipositional period and a longer ovipositional period reared on F. velutipes than on the other mushroom hosts. Every stage was different between the three Pleurotus species, except for ovipositional period and female longevity between P. ostreatus and P. eryngii var. tuoliensis. The effects of trophic levels on fecundity of T. putrescentiae are shown in Table 4. The average total eggs laid per female per day on different hosts were similar reared on A. bisporus, P. ostreatus, P. eryngii var. tuoliensis, G. lucidum, F. velutipes and A. cylindracea, but were significantly lower on A. polytricha and significantly higher on P. pulmonarius and L. edodes. The total number of eggs laid per
3.1. Development of immature stages The total developmental time of the immature stages of T. putrescentiae on different mushroom hosts at 22 C and 85% RH are shown in Table 2. Significant differences were observed among different hosts. The duration from egg to adult varied from 4.4 ± 0.2 to 9.8 ± 0.9 days (F ¼ 147.228, p < 0.05). The longest immature period occurred on Ganoderma lucidum (Karst), followed by L. edodes, A. bisporus, Agrocybe cylindracea (Maire), P. ostreatus, P. eryngii var. tuoliensis, P. pulmonarius, F. velutipes, and A. polytricha, in descending order. Egg stage was found to be the longest immature period when the mite reared on the majority of mushroom hosts except P. pulmonarius and F. velutipes, which varied from 1.0 ± 0.3 to 3.6 ± 0.9days (F ¼ 70.389, p < 0.0001). In contrary, the shortest duration was found in the tritonymph stage reared on 5 hosts and in the protonymph stage reared on 4 hosts, respectively. When the mite reared on A. polytricha and F. velutipes, the tritonymph stage was the shortest, taking less than one day, whereas
Table 3 Pre-ovipositional, ovipositional and post-ovipositional periods, female longevity of Tyrophagus putrescentiae reared on nine mushroom hosts at 22 C and 85% RH. Host
PreOviposition Post oviposition oviposition
Female longevity
L. edodes (n ¼ 27) A. bisporus (n ¼ 26) P. ostreatus (n ¼ 27) P. eryngii var.tuoliensis (n ¼ 25) P. pulmonarius (n ¼ 24) G. lucidum (n ¼ 24) A. polytricha (n ¼ 26) F. velutipes (n ¼ 24) A. cylindracea (n ¼ 24)
3.2 2.3 1.9 2.4
± ± ± ±
0.1a 0.1c 0.1d 0.4c
21.2 27.8 27.6 27.4
± ± ± ±
1.1a 0.8c 0.8c 1.1c
10.7 11.3 11.6 10.8
± ± ± ±
0.4a 0.2b 0.1b 0.8a
35.1 41.4 41.1 40.6
± ± ± ±
1.2a 0.9c 1.0c 0.3c
1.8 2.9 1.8 1.6 1.9
± ± ± ± ±
0.1d 0.1b 0.0d 0.3e 0.2d
25.1 26.8 30.8 29.8 25.5
± ± ± ± ±
2.3b 2.6c 1.1e 1.2d 2.8b
10.8 11.5 11.5 11.9 11.3
± ± ± ± ±
0.4a 0.3b 1.4b 1.0b 0.2b
37.7 41.2 44.1 43.3 38.7
± ± ± ± ±
0.4b 2.6c 2.5d 1.6d 2.8b
Values are presented as mean ± SE. n ¼ number of mites tested. Means within a column followed by the same letter are not significantly different (SNK test: P < 0.05).
Table 2 Developmental durations (days) of Tyrophagus putrescentiae reared on nine mushroom hosts at 22 C and 85% RH. Host
Stage
L. edodes (n ¼ 31) A. bisporus (n ¼ 30) P. ostreatus (n ¼ 37) P. eryngii var.tuoliensis (n ¼ 31) P. pulmonarius (n ¼ 35) G. lucidum (n ¼ 33) A. polytricha (n ¼ 32) F. velutipes (n ¼ 33) A. cylindracea (n ¼ 32)
3.6 3.0 2.6 2.3 1.7 3.4 1.8 1.0 3.2
Eggs ± ± ± ± ± ± ± ± ±
Larvae 0.9a 0.5b 0.5bc 0.2c 0.8d 0.4a 0.4d 0.3e 0.9 ab
2.7 2.3 1.7 1.3 1.2 2.1 1.4 1.7 1.6
± ± ± ± ± ± ± ± ±
0.5a 0.2b 0.5c 0.3d 0.3d 0.8b 0.4 ab 0.4c 0.4cd
Values are presented as mean ± SE. n ¼ number of mites tested. Means within a column followed by the same letter are not significantly different (SNK test: P < 0.05).
Protonymph 1.8 2.2 1.4 1.0 1.4 1.8 1.3 1.3 1.7
± ± ± ± ± ± ± ± ±
0.1b 0.1a 0.2c 0.1d 0.3c 0.4b 0.4c 0.4c 0.3b
Tritonymph 1.7 1.6 1.8 1.7 1.8 2.3 0.7 1.0 1.4
± ± ± ± ± ± ± ± ±
0.5b 0.4b 0.4b 0.5b 0.2b 0.4a 0.2e 0.3d 0.3c
Total 9.1 8.9 7.6 6.3 6.2 9.8 4.4 5.1 7.9
± ± ± ± ± ± ± ± ±
0.6b 0.6b 0.8c 0.4d 0.7d 0.9a 0.2f 0.9e 0.1c
S.-X. Qu et al. / Journal of Stored Products Research 61 (2015) 70e75 Table 4 Comparision of egg production and egg hatchability of Tyrophagus putrescentiae reared on nine mushroom hosts. Host
L. edodes (n ¼ 27) A. bisporus (n ¼ 26) P. ostreatus (n ¼ 27) P. eryngii var.tuoliensis (n ¼ 25) P. pulmonarius (n ¼ 24) G. lucidum (n ¼ 24) A. polytricha (n ¼ 26) F. velutipes (n ¼ 24) A. cylindracea (n ¼ 24)
Eggs per female per Total eggs per day* female*
Egg hatchability (%)
5.1 5.5 6.5 5.5
± ± ± ±
2.5bc 1.1 ab 1.8 ab 1.5 ab
107.4 152.0 178.7 151.1
± ± ± ±
52.4c 30.4b 47.9b 40.2b
83.4 87.4 85.0 86.4
4.1 5.8 6.9 6.1 6.0
± ± ± ± ±
1.5c 1.3 ab 2.3a 1.8 ab 1.6 ab
104.3 155.6 212.9 179.6 150.4
± ± ± ± ±
41.1c 34.0b 74.8a 53.0b 34.6b
81.3 80.5 91.5 92.8 82.2
*Values are presented as mean ± SE. n ¼ number of mites tested. Means within a column followed by the same letter are not significantly different (SNK test: P < 0.05).
female of T. putrescentiae varied from 104.3 ± 41.1 to 212.9 ± 74.8, the largest number being laid reared on A. polytricha, whereas the smallest reared on P. pulmonarius and L. edodes. There were no significant differences among the six other mushrooms. 3.3. Life-table parameters The estimated life table parameters for T. putrescentiae reared on different mushroom hosts represented in Table 5. The intrinsic rate of increase (rm) and the finite rate of increase (l) reached the maximal value were observed reared on F. velutipes followed by A. bisporus. The SNK test showed the mushroom species were more suitable for T. putrescentiae growth. The value of rm varied from 0.20 to 0.34, and l from 1.22 to 1.40. Both the maximum fecundity (212.85 eggs per female), the highest rm and l were obtained on A. polytricha followed by F. velutipe, whereas the lowest value were obtained on G. lucidum. No significant differences of life table parameters were observed between A. polytricha and F. velutipe. 3.4. Bacterial communities inhabiting mites feeding on different mushroom species A total of 30 full length sequences of 16s rRNA gene were obtained from the mites feeding on four different mushroom hosts, A. bisporus, P. eryngii var. tuoliensis, A. polytricha, F. velutipes. The changes in the diet caused by the transfer of mites from the rearing diet to the mushroom diet altered the bacteria inhabiting them (Fig. 1). The changes in the bacterial communities were influenced by the different species. The sequences showed 15 different bacterial communities after aligning the sequences using BLAST and EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/). The sequences were deposited in Genbank under Accession Nos. KF781550 e KF781566. The bacterial community inhabiting T. putrescentiae grown on the A. polytricha diet was the most diverse and included six different species (Pseudomonas, Sphingobacterium, Tistrella, Ochrobactrum, Janthinobacterium, Chryseobacterium) of which Sphingobacterium and Chryseobacterium are both associated with mites grown on the F. velutipes A. polytricha diet. Only two species were discovered inhabiting mites eating the A. bisporus diet, Serratia and Phenylobacterium. The most abundant bacteria were those closely similar to Sphingobacterium, followed by Chryseobacterium (Fig. 2). Seventeen clones were found in T. putrescentiae grown on the A. polytricha and F. velutipes test diets. However, 58.8% of these sequences were from
73
Table 5 Parameters of population increase of Tyrophagus putrescentiae reared on nine mushrooms at 22 C and 85% RH. R0
t
l
DT
Hosts
rm
L. edodes A. bisporus P. ostreatus P. eryngii var.tuoliensis P. pulmonarius G. lucidum A. polytricha F. velutipe A. cylindracea
0.20 0.21 0.23 0.25
± ± ± ±
0.0f 0.0e 0.0d 0.0c
48.4 65.4 70.3 51.7
± ± ± ±
0.3e 5.57 ± 0.0c 0.8c 5.7 ± 0.0a 0.4b 5.7 ± 0.0a 0.4d 5.3 ± 0.0e
2.38 2.2 2.25 2.1
± ± ± ±
0.0b 0.0b 0.0c 0.0d
1.2 1.2 1.4 1.4
± ± ± ±
0.0d 0.0d 0.0c 0.0b
0.21 0.20 0.34 0.32 0.23
± ± ± ± ±
0.1ef 0.0f 0.0a 0.1a 0.1d
33.0 48.9 76.9 75.5 48.7
± ± ± ± ±
0.6f 5.18 ± 0.0f 0.7e 5.5 ± 0.0b 0.4a 5.4 ± 0.0d 0.6a 5.5 ± 0.0cd 0.7e 5.4 ± 0.0e
2.3 2.3 1.8 1.8 2.3
± ± ± ± ±
0.0b 0.0a 0.0e 0.0e 0.0c
1.2 1.2 1.4 1.5 1.4
± ± ± ± ±
0.0d 0.0d 0.0a 0.0a 0.0c
Values are presented as mean ± SE. Means within a column followed by the same letter are not significantly different (SNK test: P < 0.05).
two genera. It is worth pointing out that five of seven clones from mites grown on A. bisporus belonged to Serratia marcescens after aligning the sequences using BLAST and the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/). Similarly, 99.27% with the 16S rDNA sequence (AB061685). The phylogenetic analysis showed that the bacteria inhabiting mites grown on the F. velutipes diet had a high 16S rRNA gene sequence similarity to those grown on P. eryngii var. tuoliensis, including Chryseobacterium, Corallococcus, Pedobacter, Myroides, Shinella, and Flavobacterium, while the mites grown on the A. bisporus and A. polytricha diets clustered under another group (Fig. 2).
4. Discussion In our experiments, T. putrescentiae could complete its life cycle on all tested mushroom hosts and showed significant life history differences among mushroom species. The mushroom species strongly affected the population increase, which was in agreement with the report of Kheradmand et al. (2007). The total developmental time of the immature stage was the shortest reared on A. polytricha, and was the longest reared on G. lucidum. The development time from egg to adult stage was shorter than reared on other fungi diets, such as Fusarium sp. (Nesvorn a et al., 2012) and Saccharomyces cerevisiae (Hansen) (Liu et al., 2007). Similar results for mites fed on wheat germ diets were reported by Cunnington (1976). When compared with data from previous studies on button and oyster mushrooms at 25 C, 70% RH and 16:8 (L:D) h, our study showed that T. putrescentiae had shorter developmental times and pre-ovipositional period, higher rm and longer longevity of females reared on the same mushrooms at 22 C, 85% RH and full dark. All demographic parameters were significantly different on the nine mushrooms. The intrinsic rate of increase (rm) was higher than reported earlier by Kheradmand et al. (2007). The values of rm were 0.21 and 0.23 reared on AS 2796 and Za No 3 strain used in our study, respectively (Table 5). Hubert et al. (2012) had compared the rate of development among 14 different strains of 7 Fusarium species, and discovered the rates were very significantly different among strains even the same strain was inoculated in different culture medium. Fusarium appeared to be a more suitable host for T. putrescentiae. The higher growth of mite was fed on F. verticillioides, F. poae or F. avenaceum in that study, however, was lower than two mushroom hosts, A. polytricha and F. velutipe in our study. Scientific researches have shown that different cultivars or strains of fungi hosts (differ in nutritional content) affect the
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Fig. 1. Phylogenetic assignment of bacterial 16 S rRNA gene sequences cloned from DNA extracted from Tyrophagus putrescentiae feeding on four different mushroom hosts, A. bisporus, P. eryngii var. tuoliensis, A. polytricha and F. velutipes.
Fig. 2. Phylogenetic analysis of all clones from Tyrophagus putrescentiae feeding on four different mushroom hosts, A. bisporus, P. eryngii var. tuoliensis, A. polytricha and F. velutipes.
et al., 2012). development on mites (Hubert et al., 2012; Nesvorna Cultivars or strains are variables of a predictive model. It indicates that a predictive model should include all variables, such as cultivars or stains, temperature and humidity (Hubert et al., 2013, 2014). Mushroom mycelia do not need direct light to grow, and indeed light will inhibit mycelial growth because of UV-light (Liu et al., 1990; Hou et al., 2002). One of the results of coevolution between T. putrescentiae and its fungus hosts maybe that the mite does not
need light to complete its life history. Further research is needed to confirm this hypothesis. Edible fungi are rich in protein and essential amino acids, which contain the protein accounted for about 10%e50% of the dry weight, in addition, contains carbohydrates, lipids, vitamins, minerals and other nutrients. A. polytricha contains 7.9% proteins and 64.8% carbohydrates of the dry weight, P. ostreatus contains 15.8 and 52.1%, F. velutipes contains 14.8% and 43.3%, A. bisporus contains
S.-X. Qu et al. / Journal of Stored Products Research 61 (2015) 70e75
25.0 and 44.0%, G. lucidum contains 29.1% and 7.6%, respectively (Liu et al., 1997; Zhou et al., 1998; Zhu et al., 2003). From our results, the best nutrition balance had appeared when reared on A. polytricha at 22 Cand 85% humidity. Previous study had shown that T. putrescentiae population increased by 318.5, 316.7, and 179.9 times within 6 weeks, when yeast powder, glucose, or sugar was added to the basic wheat bran diet, respectively (Huang et al., 2013). Changes in the bacterial communities inhabiting T. putrescentiae after transfer to mushroom diets were observed in our study. These results are in accord with those previously reported. Hubert et al. (2012) reported changes in the bacterial communities of this mite induced by four Fusarium fungal diets. But of 23 different bacterial communities, only one bacterium genus, Pseudomonas, was discovered in our study. These bacteria can interact with both mites and fungi and also change the palatability of fungi to the mites (Smrz et al., 1991; Smrz and Catska, 2010). Hubert et al. (2012) pointed that the changes of bacterial community associated with the storage mite might participate in the fungal mycelium hydrolysis. Our results clearly indicate that all life table parameters are affected significantly by food type. Mushroom hosts are suitability for T. putrescentiae development, especially when feeds on mycelia of A. polytricha, F. velutipe, P. ostreatus and P. eryngii var.tuoliensis. In fact, the mite infests edible mushrooms and then brings Fusarium contamination (Li, 2002). The Fusarium fungi may profit from the dispersal of spores attached on the body surface or undigested spores in the excretia (Hubert et al., 2003; Nesvorn a et al., 2012). Therefore, the control of this mite should consider the relation of mushroom hosts and other fungi. Acknowledgments This work was financially supported by the China earmarked fund for Modern Agro-industry Technology Research System (CARS 24). References Barbieri, E., Paster, B.J., Hughes, D., Zurek, L., Moser, D.P., Teske, A., Sogin, M.L., 2001. Phylogenetic characterization of epibiotic bacteria in the accessory nidamental glands and egg of the squid Loligo pealei (Cephalopoda: Loliginidae). Environ. Microbiol. 3, 151e167. Birch, L.C., 1948. The intrinsic rate of natural increase of an insect population. J. Animal Ecol. 17, 15e26. Chmielewski, W., 1999. Acceptance of buckwheat grain as food by Tyrophagus putrescentiae (Schr.) (Acari: Acaridae). Fagopyrum 16, 95e97. Cunnington, A.M., 1976. The effect of physical conditions on the development and increase of some important storage mites. Ann. Appl. Biol. 82, 175e178. Eraky, S.A., 1995. Some biological aspects of Tyrophagus putrescentiae. In: Kropcyzynska, D., Boczek, J., Tomczyk, A. (Eds.), The Acari. Oficyna Dabor, Warszawa, pp. 127e133. Franzolin, M.R., Gambale, W., Cuero, R.G., Correa, B., 1999. Interaction between toxigenic Apergillus flavus link and mites (Tyrophagus putrescentiae Schrank) on maize grains: effects on fungal growth and aflatoxin production. J. Stored Prod. Res. 35, 215e224. Hall, B.G., 2011. In: Phylogenetic Trees Made Easy: a How-to Manual. Sinauer Associates, Sunderland (MA), pp. 1e70. Hall, B.G., 2013. Building phylogenetic trees from molecular data with MEGA. Mol. Biol. Evol. 30 (5), 1229e1235. Hatcher, P.E., 1995. Three-way interactions between plant pathogenic fungi, herbivorous insects and their host plants. Biol. Rev. 70 (4), 639e694. Hou, J.R., Li, Y., Tolgor, B., Sun, X.B., 2002. Effect of UV rays on hypha growth of Morchella esculenta, Trichotoma matsutake and Collybia velutipes. 2002. J. Jilin
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Agri. Univ. 24 (6), 20e24. Huang, H., Xu, X., Lv, J., Li, G.T., Wang, E.D., Gao, Y.L., 2013. Impact of proteins and saccharides on mass production of Tyrophagus putrescentiae (Acari: Acaridae) and its predator Neoseiulus barkeri (Acari: Phytoseiidae). Biocontrol Sci. Technol. 23, 1231e1244. Hubert, J., Jarosik, V., Mourek, J., Kubatova, A., Zdarkova, E., 2004. Astigmatid mite growth and fungi preference (Acari: Acaridida): comparisons in laboratory experiments. Pedobiologia 48, 205e214. , M., Kopecký, J., 2014. The effect of Tyrophagus putrescentiae on Hubert, J., Nesvorna Fusarium poae transmission and fungal community in stored barley in a laboratory experiment. Insect Sci. 21 (1), 65e73. Hubert, J., Pekr a, S., Aulický, R., Nesvorn a, M., Stejskal, V., 2013. The effect of stored barley cultivars, temperature and humidity on population increase of Acarus siro, Lepidoglyphus destructor and Tyrophagus putrescentiae. Exp. Appl. Acarol. 60, 241e252. , M., Sa gova -Mare , M., Kopecky, Jan, 2012. Shift of bacterial Hubert, J., Nesvorna ckova community in synanthropic mite Tyrophagus putrescentiae induced by Fusarium fungal diet. PLoS One 7 (10), e48429. tov ov , E., 2003. Hubert, J., Stejskal, V., Kuba a, A., Munzbergov a, V an a, M., Zd' arkova Mites as selective fungal carriers in stored grain habitats. Exp. Appl. Acarol. 29 (1e2), 69e87. Hughes, A.M., 1976. The Mites of Stored Food and Houses. Technical Bulletin No. 9, second ed. Ministry of Agriculture, Fisheries and Food, London. Jeong, K.Y., Lee, H., Lee, J.S., Lee, J., Lee, I.Y., Ree, H.I., Yong, T.S., 2007. Molecular cloning and the allergenic characterization of tropomyosin from Tyrophagus putrescentiae. Protein Pept. Lett. 14, 431e436. Kheradmand, K., Kamali, K., Fathipour, Y., Mohammadi Goltapeh, E., 2007. Development, life table and thermal requirement of Tyrophagus putrescentiae (Astigmata: Acaridae) on mushrooms. J. Stored Prod. Res. 43, 276e281. Li, C.P., 2002. Experiment study on the capacity of Tyrophagus putrescentiae in carrying and transmitting mold. Acta Arachnol. Sin. 11 (1), 58e60. Li, C.P., Cui, Y.B., Wang, J., Yang, Q.G., Tian, Y., 2003. Acaroid mite, intestinal and urinary acariasis. World J. Gastroenterol. 9 (4), 874e877. Liu, T., Jin, D.C., Guo, J.J., 2007. Laboratory population life table of Tyrophagus putrescentiae at different temperatures. Plant Prot. 33 (3), 68e72. Liu, Z.L., Han, C.P., Wei, L.L., 1990. Fungicidal effect of ultraviolet radiation. Chin. J. Disinfect. 7 (2), 95e97. Liu, Q., Teng, W., Su, B.Y., 1997. Analysis of nutritive value of 9 species of edible fungi in China. Amino Acids Biotic Resour. 19 (2), 7e14. Lu, M., Li, Y., 2012. New development trend of edible fungus industry in China. J. Anhui Agric. Sci. 40 (5), 3121e3224. Lu, Y.H., 2002. The biological characteristics and control countermeasures of important edible mushroom mite e Tyrophagus putrescentiae. J. Anhui Agric. Sci. 30 (1), 100e101. Moran, N.A., 2002. The ubiquitous and varied role of infection in the lives of animals and plants. Am. Nat. 160, S1eS8. Nesvorn a, M., Gabrielova, L., Hubert, J., 2012. Tyrophagus putrescentiae is able to graze and develop on Fusarium fungi of mycotoxins importance under laboratory conditions. J. Stored Prod. Res. 48, 37e45. Roy, M., Brodeur, J., Cloutier, C., 2002. Relationship between temperature and developmental rate of Stethorus punctillum (Coleoptera: Coccinellidae) and its prey Tetranychus mcdanieli (Acarina: Tetranychidae). Environ. Entomol. 31, 177e187. Smrz, J., Catska, V., 2010. Mycophagous mites and their internal associated bacteria cooperate to digest chitin in soil. Symbiosis 52, 33e40. Smrz, J., Catska, V., 1989. The effect of the consumption of some soil fungi on the internal microanatomy of the mite Tyrophagus putrescentiae (Schrank) (Acari: Acaridida). Acta Univ. Carolinae-Biologica 33, 81e93. Smrz, J., Svobodova, J., Catska, V., 1991. Synergetic participation of Tyrophagus putrescentiae (Schrank) (Acari, Acaridida) and its associated bacteria on the destruction of some soil micromycetes. J. Appl. Entomol. 111, 206e210. Wu, D., Daugherty, S.C., Van Aken, S.E., Pai, G.H., Watkins, K.L., Khouri, H., Eisen, J.A., 2006. Metabolic complementarity and genomics of the dual bacterial symbiosis of sharp shooters. PLoS Biol. 4, e188. Zhang, G.L., Jin, L.Z., 1960. Species and distribution of stored product mites in China. Food Sci. Technol. Commun. 2, 23e24. Zhang, Y.X., Lin, J.Z., Huang, J.H., Zhu, P.G., 1992. Studies on Tyrophagus putreseentiae (Schrank) and its damage to the Edible Fungi. J. Fujian Agric. Sci. 7 (2), 91e94. Zhou, X.W., Lin, J., Zhou, L., 1998. Determine and analysis on primary nutritional components of Ganoderma spp. J. Shanxi Normal Univ. Nat. Sci. Ed. 26, 214e217. Zhu, X.P., Liu, W., Gao, S.G., Hou, D.J., 2003. Determination and analysis of total nitrogen, protein and lysine contents in several Pleurotus ostreatus and Flammulina velutipes strains. J. Hebei Vocation Tech. Teach. Coll. 17 (4), 34e36. Zou, P., 1987. The introduction of common species of mites in mushroom. Edible fungi 3, 29.
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Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
Effects of different edible mushroom hosts on the development, reproduction and bacterial community of Tyrophagus putrescentiae (Schrank) S.-X. Qu a, b, H.-P. Li b, L. Ma b, L.-J. Hou b, J.-S. Lin b, J.-D. Song b, X.-Y. Hong a, * a b
Department of Entomology, Nanjing Agricultural University, Nanjing 210095, China Institute of Vegetable Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 March 2014 Received in revised form 27 November 2014 Accepted 12 December 2014 Available online 19 December 2014
The storage mite, Tyrophagus putrescentiae (Schrank), infests various stored products, plant seeds, chinese herbal medicines and edible mushrooms in China. To assess interactions between edible mushroom hosts and the mite, we investigated the effects of nine species on development and reproduction at 22 Cand 85% humidity in the laboratory. We also demonstrated changes in the bacterial community of T. putrescentiae reared on four mushroom hosts. All the immature stages, female longevity and reproductive periods were significantly affected by the mushroom species, total immature developmental time varied from 4.41 ± 0.20 (reared on Auricularia polytricha) to 9.79 ± 0.92 days (when reared on Ganoderma lucidum). The egg stage was the longest period of the immature stages, whereas the protonymph and tritonymph stages were the shortest at about 2 days. The net reproductive rate (R0), and the intrinsic rate of increase (rm) were significantly different on the nine mushroom hosts. Both the maximum fecundity (212.85 eggs per female) and the highest rm were obtained on A. polytricha. The results indicated that T. putrescentiae had a wide range of adaptability to edible mushrooms, with development and reproduction of the mite being significantly affected by different species. Changes in the bacterial communities inhabiting the mites were influenced by mushroom hosts. A total of 30 full length sequences of 16s rRNA gene were obtained from the mite reared on four hosts. The bacterial community inhabiting T. putrescentiae on A. polytricha host was the most diverse with six different species, two of these genera occurred on Flammulina velutipes host. Consequently the interactions between T. putrescentiae and mushroom hosts can influence both the biological characteristics and bacterial community. The changes of bacterial community might participate in the different fungal mycelium hydrolysis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Mushroom pest Trophic levels Storage mite Flour mite
1. Introduction Tyrophagus putrescentiae (Schrank) belongs to the class Arachnida (Acarina, acaridida, Acaridae, Tyrophagus). It is one of the most common synanthropic mites with a cosmopolitan occurrence, and is a major pest of stored grain. It attacks a very wide range of produce including rice, wheat bran, herbs, cheese, nuts and candied fruit, peanut, and flour, as well as edible fungi and mushroom beds (Zhang and Jin, 1960; Hughes, 1976; Zou, 1987; Eraky, 1995; Chmielewski, 1999). Its feeding on fungi has been widely reported
* Corresponding author. E-mail address: [email protected] (X.-Y. Hong). http://dx.doi.org/10.1016/j.jspr.2014.12.003 0022-474X/© 2014 Elsevier Ltd. All rights reserved.
and it can develop on many fungal species (Smrz and Catska, 1989; Zhang et al., 1992; Hubert et al., 2004). The mite can also promote fungal growth by removing senescent parts of the mycelium which stimulates fungal metabolism (Hatcher, 1995; Hubert et al., 2003). T. putrescentiae is able to transfer Aspergillus flavus (Link) to sterile grain, resulting in fungus growth and aflatoxin production (Franzolin et al., 1999). The latest study had shown that the mite transmited an amycotoxin-producing strain of Fusarium poae (Peck) Wollenw to stored barley, and increased the deoxynivalenol (DON) content in the barley following successful F. poae transfer (Hubert et al., 2014). With such a wide range of hosts and suitable habitats, T. putrescentiae is a major pest of mushroom cultivation and storage (Lu, 2002). According to statistics, its mushroom hosts include
S.-X. Qu et al. / Journal of Stored Products Research 61 (2015) 70e75
Lentinula edodes (Berk), Agaricus bisporus (Lange), Pleurotus ostreatus (Kumm), Ganoderma sp., and Pleurotus pulmonarius (Fr.). It can carry and spread pathogens, reduce mushroom production, cause dried mushroom mildew, and cause human allergic dermatitis, pulmonary acariasis, and intestinal acariasis (Li et al., 2003; Jeong et al., 2007), seriously affecting the economic efficiency of mushroom production and the health of mushroom farmers. According to the Ministry of Agriculture, Chinese edible mushroom output has reached more than 10 million tons annually in recent years, accounting for over 70% of the total global production. As many as 50 varieties of edible mushroom are cultivated in China, 20 million people are engaged in the industry (Lu and Li, 2012). So far, only one study has reported the effects of temperature and edible fungi on development, life table, and thermal requirements of T. putrescentiae. The net reproductive rate (R0), and the intrinsic rate of increase (rm), were significantly different on the two mushroom hosts (Kheradmand et al., 2007). Bacteria can play beneficial, and often essential, roles in the lives of their hosts. In animals that carry vertically transmitted, intracellular bacteria, the host and its symbiont community form an inseparable holobiont with a shared metabolism and evolutionary fate (Wu et al., 2006; Moran, 2002). The aim of this study is to determine the effects of nine largest cultivated mushroom varieties on the development and reproduction of T. putrescentiae at 22 C and 85% humidity in the laboratory, and to demonstrate changes in the bacterial community inhabiting T. putrescentiae induced by different mushroom hosts.
2.1. Mites and mushroom culture The storage mites, T. putrescentiae used in the study were supplied from stock cultures originating from a population collected in 2012 on wood ear mushroom Auricularia polytricha (Mont.) in Feng County (Jiangsu, China), which had been kept in the laboratory at 25 C and approximately 85% RH. The mites used to assess host suitability were initially fed the nine edible mushrooms to be tested for four generations, to acclimatize them to each host. One week before the start of the experiments, the mushroom mycelia were inoculated onto potato dextrose agar (PDA) plates in 50 mm diameter Petri dishes. The dishes were covered with lids and placed in a growth chamber at
Table 1 List of edible mushroom species and strains from China used to investigate development, reproduction and bacterial communities in Tyrophagus putrescentiae. Species
Stain no.
Lentinula edodes Wuxiang No1 Agaricus bisporus AS2796 Pleurotus Za No 3 ostreatus P. eryngii Zhongnong var.tuoliensis No1 P. pulmonarius 206 Ganoderma lucidum Auricularia polytricha Flammulina velutipes Agrocybe cylindracea
Family and genus
Agaricales, Tricholomataceae, Lentinula Agaricales, Agaricaceae, Agaricus Agaricales, pleurotaceae, Pleurotus Agaricales, pleurotaceae, Pleurotus Agaricales, pleurotaceae, Pleurotus Taishan Aphyllophorales,Polyporaceae, Ganoderma Fengmao Auriculariales, Auriculariaceace, No 3 Auricularia Danbaijin Agaricales, Tricholomataceae, Flammulina Gucha No 1 Agaricales, Bolbitiaceae, Agrocybe
25 C and 60% RH in complete darkness. The mushroom species and strains used in the experiments are described in Table 1.
2.2. Development, reproduction and longevity Following inoculation of the nine mushroom mycelia onto the 50 mm PDA Petri dishes, a mating pair of mites was placed on each of the nine edible mushroom substrates. Ten replicates per mushroom species were used to determine the durations of the developmental stages of the mite lifecycle. Newly-laid (one-day old) eggs were separated with a hair brush onto five new 50 mm Petri dishes with the corresponding host mushroom mycelia using an S8APO dissection microscope (Leica, Germany). Each Petri dish received 5e7 eggs of the same age. Petri dishes were covered with Parafilm® (Bemis, USA) wrap and put into an SPX-2501C type artificial climate box (Suzhou Jiangdong Precision Instrument Co., Ltd.). All tests were performed in the laboratory at 22 ± 1 C, 85% ± 5% RH, in the dark. Observations of mite mortality and fecundity were made every day at 9:00 am and 15:00 pm.
2.3. Life-table parameters A life table was constructed from the observed survival and fecundity rates for individuals. Parameters were calculated by the methods of Birch (1948) and Roy et al. (2002). The intrinsic rate of increase (rm) was estimated by nonlinear regression according to:
X ½expð rm xÞlx mx ¼ 1
2. Materials and methods
Province of origin Zhejiang Fujiang Jiangsu
71
(1)
where rm is the intrinsic rate of increase, x is female age in days, lx is the fraction of females surviving to age x (age-specific survival rate), and mx is the expected number of daughters produced per female alive at age x (age-specific fecundity rate), obtained by multiplying the number of eggs by the age-specific sex ratio, which is defined as the proportion of females in the progeny. The net reproductive rate (R0), the mean generation time (t) in days, the finite rate of increase (l) and the doubling time (DT) were computed by the respective equations:
R0 ¼
X
lx mx
(2)
t ¼ ln R0 0=rm
(3)
l ¼ e rm
(4)
DT ¼ ln2=rm
(5)
One-way ANOVA was used to evaluate the effect of mushrooms hosts on developmental times, reproductive periods, female longevity and fecundity of T. putrescentiae. Means were compared by the SNK test at the 5% level using. These analyses were performed with SPSS 20.0.
Beijing Jiangsu
2.4. Bacterial communities inhabiting mites feeding on different edible mushroom species
Shandong Jiangsu Jiangsu Fujian
In this experiment, we used four different populations of the storage mite feeding on four mushroom species, A. bisporus, Pleurotus eryngii var. tuoliensis (Mou), A. polytricha, Flammulina velutipes (Fr.) to evaluate differences in their associated bacterial communities. Bacterial communities were characterized by 16S rRNA gene sequencing.
72
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2.4.1. DNA extraction, PCR amplification and sequencing Genomic DNA was extracted by homogenizing a single female adult using the Wizard®SV Genomic DNA Purification System (Promega, USA).The 16S rRNA gene was amplified using PCR, performed with the universal primers UF: 5-AGA GTT TGA TYM TGGC 3 (position 8e23) and UR: 5-GYT ACC TTG TTA CGA CTT (position 1496e1514) using a MyCycler™ Thermal Cycler (Bio-Rad, USA) (Barbieri et al., 2001). A total volume of 50 mL PCR reaction mixture contained 25 mL GoTaq® Green Master Mix (Promega, USA), 5 mL each of forward and reverse primers, and 3 mL of template DNA. Reactions were cycled 30 times at 94 C for 30 s, 50 C for 90 s, and 72 C for 1 min. The PCR products were electrophoresed on a 1.0% agarose gel in TBE for 20 min at 100 mA, and then purified with the AxyPrep™ PCR Clean-Up Kit (Axygen, USA). The PCR products were cloned using the pGEM-T easy vector system (Promega, USA) and transformed into Escherichia coli DH5a, and selected colonies from each plate were picked at random and sequenced by Sangon, Shanghai. 2.4.2. Phylogenetic analysis An initial alignment of all 16S rRNA gene sequences was performed using MEGA 5.1 (Hall, 2013). The Neighbor-Joining method was used based on the Jukes-Cantor distance matrix (1000 bootstrap resamplings) in MEGA 5.1, and the maximum-likelihood method with the Jones Taylor Thornton (JTT) model (1000 bootstrap resamplings). The resulting phylograms were finalized using MEGA 5.1 (Hall, 2011). 3. Results
other hosts needed at least 1e2 days to complete this stage. In Pleurotus, there were differences in the immature stages, except for the tritonymph stage. No differences were found in larvae, tritonymph and the total developmental time between P. eryngii var. tuoliensis and P. pulmonarius. 3.2. Longevity and reproductive period Females reared on different mushrooms were tested for the duration of their pre-ovipositional, ovipositional and postovipositional periods, and longevity (Table 3). The total longevity of females ranged from 35.1 ± 1.2 to 44.1 ± 2.5 days. Significant differences were observed among the nine hosts. The variation in post-ovipositional period reared on the nine different mushrooms was lustered into two groups without significant differences within them (F ¼ 9.620, p < 0.0001): among L. edodes, P. eryngii var. tuoliensis and P. pulmonarius; and among A. bisporus, P. ostreatus, G. lucidum, A. polytricha, F. velutipes, and A. cylindracea. Females had a shorter pre-ovipositional period and a longer ovipositional period reared on F. velutipes than on the other mushroom hosts. Every stage was different between the three Pleurotus species, except for ovipositional period and female longevity between P. ostreatus and P. eryngii var. tuoliensis. The effects of trophic levels on fecundity of T. putrescentiae are shown in Table 4. The average total eggs laid per female per day on different hosts were similar reared on A. bisporus, P. ostreatus, P. eryngii var. tuoliensis, G. lucidum, F. velutipes and A. cylindracea, but were significantly lower on A. polytricha and significantly higher on P. pulmonarius and L. edodes. The total number of eggs laid per
3.1. Development of immature stages The total developmental time of the immature stages of T. putrescentiae on different mushroom hosts at 22 C and 85% RH are shown in Table 2. Significant differences were observed among different hosts. The duration from egg to adult varied from 4.4 ± 0.2 to 9.8 ± 0.9 days (F ¼ 147.228, p < 0.05). The longest immature period occurred on Ganoderma lucidum (Karst), followed by L. edodes, A. bisporus, Agrocybe cylindracea (Maire), P. ostreatus, P. eryngii var. tuoliensis, P. pulmonarius, F. velutipes, and A. polytricha, in descending order. Egg stage was found to be the longest immature period when the mite reared on the majority of mushroom hosts except P. pulmonarius and F. velutipes, which varied from 1.0 ± 0.3 to 3.6 ± 0.9days (F ¼ 70.389, p < 0.0001). In contrary, the shortest duration was found in the tritonymph stage reared on 5 hosts and in the protonymph stage reared on 4 hosts, respectively. When the mite reared on A. polytricha and F. velutipes, the tritonymph stage was the shortest, taking less than one day, whereas
Table 3 Pre-ovipositional, ovipositional and post-ovipositional periods, female longevity of Tyrophagus putrescentiae reared on nine mushroom hosts at 22 C and 85% RH. Host
PreOviposition Post oviposition oviposition
Female longevity
L. edodes (n ¼ 27) A. bisporus (n ¼ 26) P. ostreatus (n ¼ 27) P. eryngii var.tuoliensis (n ¼ 25) P. pulmonarius (n ¼ 24) G. lucidum (n ¼ 24) A. polytricha (n ¼ 26) F. velutipes (n ¼ 24) A. cylindracea (n ¼ 24)
3.2 2.3 1.9 2.4
± ± ± ±
0.1a 0.1c 0.1d 0.4c
21.2 27.8 27.6 27.4
± ± ± ±
1.1a 0.8c 0.8c 1.1c
10.7 11.3 11.6 10.8
± ± ± ±
0.4a 0.2b 0.1b 0.8a
35.1 41.4 41.1 40.6
± ± ± ±
1.2a 0.9c 1.0c 0.3c
1.8 2.9 1.8 1.6 1.9
± ± ± ± ±
0.1d 0.1b 0.0d 0.3e 0.2d
25.1 26.8 30.8 29.8 25.5
± ± ± ± ±
2.3b 2.6c 1.1e 1.2d 2.8b
10.8 11.5 11.5 11.9 11.3
± ± ± ± ±
0.4a 0.3b 1.4b 1.0b 0.2b
37.7 41.2 44.1 43.3 38.7
± ± ± ± ±
0.4b 2.6c 2.5d 1.6d 2.8b
Values are presented as mean ± SE. n ¼ number of mites tested. Means within a column followed by the same letter are not significantly different (SNK test: P < 0.05).
Table 2 Developmental durations (days) of Tyrophagus putrescentiae reared on nine mushroom hosts at 22 C and 85% RH. Host
Stage
L. edodes (n ¼ 31) A. bisporus (n ¼ 30) P. ostreatus (n ¼ 37) P. eryngii var.tuoliensis (n ¼ 31) P. pulmonarius (n ¼ 35) G. lucidum (n ¼ 33) A. polytricha (n ¼ 32) F. velutipes (n ¼ 33) A. cylindracea (n ¼ 32)
3.6 3.0 2.6 2.3 1.7 3.4 1.8 1.0 3.2
Eggs ± ± ± ± ± ± ± ± ±
Larvae 0.9a 0.5b 0.5bc 0.2c 0.8d 0.4a 0.4d 0.3e 0.9 ab
2.7 2.3 1.7 1.3 1.2 2.1 1.4 1.7 1.6
± ± ± ± ± ± ± ± ±
0.5a 0.2b 0.5c 0.3d 0.3d 0.8b 0.4 ab 0.4c 0.4cd
Values are presented as mean ± SE. n ¼ number of mites tested. Means within a column followed by the same letter are not significantly different (SNK test: P < 0.05).
Protonymph 1.8 2.2 1.4 1.0 1.4 1.8 1.3 1.3 1.7
± ± ± ± ± ± ± ± ±
0.1b 0.1a 0.2c 0.1d 0.3c 0.4b 0.4c 0.4c 0.3b
Tritonymph 1.7 1.6 1.8 1.7 1.8 2.3 0.7 1.0 1.4
± ± ± ± ± ± ± ± ±
0.5b 0.4b 0.4b 0.5b 0.2b 0.4a 0.2e 0.3d 0.3c
Total 9.1 8.9 7.6 6.3 6.2 9.8 4.4 5.1 7.9
± ± ± ± ± ± ± ± ±
0.6b 0.6b 0.8c 0.4d 0.7d 0.9a 0.2f 0.9e 0.1c
S.-X. Qu et al. / Journal of Stored Products Research 61 (2015) 70e75 Table 4 Comparision of egg production and egg hatchability of Tyrophagus putrescentiae reared on nine mushroom hosts. Host
L. edodes (n ¼ 27) A. bisporus (n ¼ 26) P. ostreatus (n ¼ 27) P. eryngii var.tuoliensis (n ¼ 25) P. pulmonarius (n ¼ 24) G. lucidum (n ¼ 24) A. polytricha (n ¼ 26) F. velutipes (n ¼ 24) A. cylindracea (n ¼ 24)
Eggs per female per Total eggs per day* female*
Egg hatchability (%)
5.1 5.5 6.5 5.5
± ± ± ±
2.5bc 1.1 ab 1.8 ab 1.5 ab
107.4 152.0 178.7 151.1
± ± ± ±
52.4c 30.4b 47.9b 40.2b
83.4 87.4 85.0 86.4
4.1 5.8 6.9 6.1 6.0
± ± ± ± ±
1.5c 1.3 ab 2.3a 1.8 ab 1.6 ab
104.3 155.6 212.9 179.6 150.4
± ± ± ± ±
41.1c 34.0b 74.8a 53.0b 34.6b
81.3 80.5 91.5 92.8 82.2
*Values are presented as mean ± SE. n ¼ number of mites tested. Means within a column followed by the same letter are not significantly different (SNK test: P < 0.05).
female of T. putrescentiae varied from 104.3 ± 41.1 to 212.9 ± 74.8, the largest number being laid reared on A. polytricha, whereas the smallest reared on P. pulmonarius and L. edodes. There were no significant differences among the six other mushrooms. 3.3. Life-table parameters The estimated life table parameters for T. putrescentiae reared on different mushroom hosts represented in Table 5. The intrinsic rate of increase (rm) and the finite rate of increase (l) reached the maximal value were observed reared on F. velutipes followed by A. bisporus. The SNK test showed the mushroom species were more suitable for T. putrescentiae growth. The value of rm varied from 0.20 to 0.34, and l from 1.22 to 1.40. Both the maximum fecundity (212.85 eggs per female), the highest rm and l were obtained on A. polytricha followed by F. velutipe, whereas the lowest value were obtained on G. lucidum. No significant differences of life table parameters were observed between A. polytricha and F. velutipe. 3.4. Bacterial communities inhabiting mites feeding on different mushroom species A total of 30 full length sequences of 16s rRNA gene were obtained from the mites feeding on four different mushroom hosts, A. bisporus, P. eryngii var. tuoliensis, A. polytricha, F. velutipes. The changes in the diet caused by the transfer of mites from the rearing diet to the mushroom diet altered the bacteria inhabiting them (Fig. 1). The changes in the bacterial communities were influenced by the different species. The sequences showed 15 different bacterial communities after aligning the sequences using BLAST and EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/). The sequences were deposited in Genbank under Accession Nos. KF781550 e KF781566. The bacterial community inhabiting T. putrescentiae grown on the A. polytricha diet was the most diverse and included six different species (Pseudomonas, Sphingobacterium, Tistrella, Ochrobactrum, Janthinobacterium, Chryseobacterium) of which Sphingobacterium and Chryseobacterium are both associated with mites grown on the F. velutipes A. polytricha diet. Only two species were discovered inhabiting mites eating the A. bisporus diet, Serratia and Phenylobacterium. The most abundant bacteria were those closely similar to Sphingobacterium, followed by Chryseobacterium (Fig. 2). Seventeen clones were found in T. putrescentiae grown on the A. polytricha and F. velutipes test diets. However, 58.8% of these sequences were from
73
Table 5 Parameters of population increase of Tyrophagus putrescentiae reared on nine mushrooms at 22 C and 85% RH. R0
t
l
DT
Hosts
rm
L. edodes A. bisporus P. ostreatus P. eryngii var.tuoliensis P. pulmonarius G. lucidum A. polytricha F. velutipe A. cylindracea
0.20 0.21 0.23 0.25
± ± ± ±
0.0f 0.0e 0.0d 0.0c
48.4 65.4 70.3 51.7
± ± ± ±
0.3e 5.57 ± 0.0c 0.8c 5.7 ± 0.0a 0.4b 5.7 ± 0.0a 0.4d 5.3 ± 0.0e
2.38 2.2 2.25 2.1
± ± ± ±
0.0b 0.0b 0.0c 0.0d
1.2 1.2 1.4 1.4
± ± ± ±
0.0d 0.0d 0.0c 0.0b
0.21 0.20 0.34 0.32 0.23
± ± ± ± ±
0.1ef 0.0f 0.0a 0.1a 0.1d
33.0 48.9 76.9 75.5 48.7
± ± ± ± ±
0.6f 5.18 ± 0.0f 0.7e 5.5 ± 0.0b 0.4a 5.4 ± 0.0d 0.6a 5.5 ± 0.0cd 0.7e 5.4 ± 0.0e
2.3 2.3 1.8 1.8 2.3
± ± ± ± ±
0.0b 0.0a 0.0e 0.0e 0.0c
1.2 1.2 1.4 1.5 1.4
± ± ± ± ±
0.0d 0.0d 0.0a 0.0a 0.0c
Values are presented as mean ± SE. Means within a column followed by the same letter are not significantly different (SNK test: P < 0.05).
two genera. It is worth pointing out that five of seven clones from mites grown on A. bisporus belonged to Serratia marcescens after aligning the sequences using BLAST and the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/). Similarly, 99.27% with the 16S rDNA sequence (AB061685). The phylogenetic analysis showed that the bacteria inhabiting mites grown on the F. velutipes diet had a high 16S rRNA gene sequence similarity to those grown on P. eryngii var. tuoliensis, including Chryseobacterium, Corallococcus, Pedobacter, Myroides, Shinella, and Flavobacterium, while the mites grown on the A. bisporus and A. polytricha diets clustered under another group (Fig. 2).
4. Discussion In our experiments, T. putrescentiae could complete its life cycle on all tested mushroom hosts and showed significant life history differences among mushroom species. The mushroom species strongly affected the population increase, which was in agreement with the report of Kheradmand et al. (2007). The total developmental time of the immature stage was the shortest reared on A. polytricha, and was the longest reared on G. lucidum. The development time from egg to adult stage was shorter than reared on other fungi diets, such as Fusarium sp. (Nesvorn a et al., 2012) and Saccharomyces cerevisiae (Hansen) (Liu et al., 2007). Similar results for mites fed on wheat germ diets were reported by Cunnington (1976). When compared with data from previous studies on button and oyster mushrooms at 25 C, 70% RH and 16:8 (L:D) h, our study showed that T. putrescentiae had shorter developmental times and pre-ovipositional period, higher rm and longer longevity of females reared on the same mushrooms at 22 C, 85% RH and full dark. All demographic parameters were significantly different on the nine mushrooms. The intrinsic rate of increase (rm) was higher than reported earlier by Kheradmand et al. (2007). The values of rm were 0.21 and 0.23 reared on AS 2796 and Za No 3 strain used in our study, respectively (Table 5). Hubert et al. (2012) had compared the rate of development among 14 different strains of 7 Fusarium species, and discovered the rates were very significantly different among strains even the same strain was inoculated in different culture medium. Fusarium appeared to be a more suitable host for T. putrescentiae. The higher growth of mite was fed on F. verticillioides, F. poae or F. avenaceum in that study, however, was lower than two mushroom hosts, A. polytricha and F. velutipe in our study. Scientific researches have shown that different cultivars or strains of fungi hosts (differ in nutritional content) affect the
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S.-X. Qu et al. / Journal of Stored Products Research 61 (2015) 70e75
Fig. 1. Phylogenetic assignment of bacterial 16 S rRNA gene sequences cloned from DNA extracted from Tyrophagus putrescentiae feeding on four different mushroom hosts, A. bisporus, P. eryngii var. tuoliensis, A. polytricha and F. velutipes.
Fig. 2. Phylogenetic analysis of all clones from Tyrophagus putrescentiae feeding on four different mushroom hosts, A. bisporus, P. eryngii var. tuoliensis, A. polytricha and F. velutipes.
et al., 2012). development on mites (Hubert et al., 2012; Nesvorna Cultivars or strains are variables of a predictive model. It indicates that a predictive model should include all variables, such as cultivars or stains, temperature and humidity (Hubert et al., 2013, 2014). Mushroom mycelia do not need direct light to grow, and indeed light will inhibit mycelial growth because of UV-light (Liu et al., 1990; Hou et al., 2002). One of the results of coevolution between T. putrescentiae and its fungus hosts maybe that the mite does not
need light to complete its life history. Further research is needed to confirm this hypothesis. Edible fungi are rich in protein and essential amino acids, which contain the protein accounted for about 10%e50% of the dry weight, in addition, contains carbohydrates, lipids, vitamins, minerals and other nutrients. A. polytricha contains 7.9% proteins and 64.8% carbohydrates of the dry weight, P. ostreatus contains 15.8 and 52.1%, F. velutipes contains 14.8% and 43.3%, A. bisporus contains
S.-X. Qu et al. / Journal of Stored Products Research 61 (2015) 70e75
25.0 and 44.0%, G. lucidum contains 29.1% and 7.6%, respectively (Liu et al., 1997; Zhou et al., 1998; Zhu et al., 2003). From our results, the best nutrition balance had appeared when reared on A. polytricha at 22 Cand 85% humidity. Previous study had shown that T. putrescentiae population increased by 318.5, 316.7, and 179.9 times within 6 weeks, when yeast powder, glucose, or sugar was added to the basic wheat bran diet, respectively (Huang et al., 2013). Changes in the bacterial communities inhabiting T. putrescentiae after transfer to mushroom diets were observed in our study. These results are in accord with those previously reported. Hubert et al. (2012) reported changes in the bacterial communities of this mite induced by four Fusarium fungal diets. But of 23 different bacterial communities, only one bacterium genus, Pseudomonas, was discovered in our study. These bacteria can interact with both mites and fungi and also change the palatability of fungi to the mites (Smrz et al., 1991; Smrz and Catska, 2010). Hubert et al. (2012) pointed that the changes of bacterial community associated with the storage mite might participate in the fungal mycelium hydrolysis. Our results clearly indicate that all life table parameters are affected significantly by food type. Mushroom hosts are suitability for T. putrescentiae development, especially when feeds on mycelia of A. polytricha, F. velutipe, P. ostreatus and P. eryngii var.tuoliensis. In fact, the mite infests edible mushrooms and then brings Fusarium contamination (Li, 2002). The Fusarium fungi may profit from the dispersal of spores attached on the body surface or undigested spores in the excretia (Hubert et al., 2003; Nesvorn a et al., 2012). Therefore, the control of this mite should consider the relation of mushroom hosts and other fungi. Acknowledgments This work was financially supported by the China earmarked fund for Modern Agro-industry Technology Research System (CARS 24). References Barbieri, E., Paster, B.J., Hughes, D., Zurek, L., Moser, D.P., Teske, A., Sogin, M.L., 2001. Phylogenetic characterization of epibiotic bacteria in the accessory nidamental glands and egg of the squid Loligo pealei (Cephalopoda: Loliginidae). Environ. Microbiol. 3, 151e167. Birch, L.C., 1948. The intrinsic rate of natural increase of an insect population. J. Animal Ecol. 17, 15e26. Chmielewski, W., 1999. Acceptance of buckwheat grain as food by Tyrophagus putrescentiae (Schr.) (Acari: Acaridae). Fagopyrum 16, 95e97. Cunnington, A.M., 1976. The effect of physical conditions on the development and increase of some important storage mites. Ann. Appl. Biol. 82, 175e178. Eraky, S.A., 1995. Some biological aspects of Tyrophagus putrescentiae. In: Kropcyzynska, D., Boczek, J., Tomczyk, A. (Eds.), The Acari. Oficyna Dabor, Warszawa, pp. 127e133. Franzolin, M.R., Gambale, W., Cuero, R.G., Correa, B., 1999. Interaction between toxigenic Apergillus flavus link and mites (Tyrophagus putrescentiae Schrank) on maize grains: effects on fungal growth and aflatoxin production. J. Stored Prod. Res. 35, 215e224. Hall, B.G., 2011. In: Phylogenetic Trees Made Easy: a How-to Manual. Sinauer Associates, Sunderland (MA), pp. 1e70. Hall, B.G., 2013. Building phylogenetic trees from molecular data with MEGA. Mol. Biol. Evol. 30 (5), 1229e1235. Hatcher, P.E., 1995. Three-way interactions between plant pathogenic fungi, herbivorous insects and their host plants. Biol. Rev. 70 (4), 639e694. Hou, J.R., Li, Y., Tolgor, B., Sun, X.B., 2002. Effect of UV rays on hypha growth of Morchella esculenta, Trichotoma matsutake and Collybia velutipes. 2002. J. Jilin
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