Supplementary Material

Journal: Naturewissenchaften

Species-specific and female host-biased

ectophoresy in the roundworm

Caenorhabditis japonica

Toyoshi Yoshiga1*, Yuji Ishikawa1, Ryusei Tanaka1, Mantaro Hironaka2, Etsuko Okumura1

1Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan

2Department of Biology, Faculty of Medicine, Hamamatsu University School of Medicine

*Corresponding author:

email:

phone & fax: +81-952-288746

Supplementary Material

Morphological and molecular identification of nematodes

DL recovered from P. japonensiswere placed on NGM pre-seeded with Escherichia coli OP50 (Sulston and Hodgkin 1988), and adult nematodes that developed from DL were examined morphologically. To confirm the species identity, in addition to morphological observations, 10 of 35 DL from the thorax region and seven of seven DL from the abdominal regionof a single adult female insect from Takeo, as well as another seven DL from seven adult female insects fromKanzaki (Table 1 in supplementary material)were individually subjected to DNA analysis.

DNA was extracted from a single DL by crushing it with a small piece of filter paper (Iwahori et al. 2000) and transferring it to 25 µl of worm lysis buffer (50 mMKCl; 10 mMTris-HCl, pH 8.2; 2.5 mM MgCl2; 0.45% Tween 20; 0.05% gelatin; and 60 µg/ml proteinase K; modified from Joyce et al. 1994). This solution was incubated at 65°C for 1 h, and then at 95°C for 15 min. After centrifugation at 4,000 × g for 2 min, the supernatant containing nematode DNA was collected and stored at −30°C until use. The 5′ end of the nematode SSU rDNA of nematodes was amplified by polymerase chain reaction (PCR) using the primers SSU18A and SSU26R (Blaxter et al. 1998) in a 25 µl reaction mixture containing 2.5 µl 10× PCR buffer, 2 µl dNTP mixture, 0.125 µl Ex Taq (Takara Co., Ltd.), 1 µM of each primer, and 2 µl template DNA solution. The PCR reaction mixture was denatured at 95°C for 3 min followed by 35 cycles of 95°C for 30 sec, 45°C for 30 sec, and 72°C for 1 min, followed by a post-amplification extension at 72°C for 8 min. The PCR products were separated on a 1% agarose gel. A DNA band of approximately 850 bp was excised from the gel, and the DNA was purified using the Gel-M Gel Extraction System (Viogene). Purified DNA was prepared for direct sequencing using one of the primers 22F, 9FX, 22R, or 9R (Blaxter et al. 1998), and the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing kit. Sequencing was performed using an ABI PRISM® 310 Genetic Analyzer.

Detection of C. japonica from invertebrates

Invertebrates from the Hinokuma Mountain Park, where P. japonensis live, were collected and sacrificed to detect C. japonica. In September 2009, approximately 11 kg of soil sample (litter and thin cambisol under the litter) was collected from a plot (5 m × 5 m) under the S. jasminodora tree where severalfruits, some nests of P. japonensis, and several P. japonensisnymphs feeding on the fruits were observed.Sampling was conducted in Septemberwhen the P. japonensis nymph cadavers and the S. jasminodora fruits, a possible nutrient source for C. japonica propagation, were no longer available on the litter to avoid accidental associations during sampling. To collect invertebrates wandering the plot area, 12 pitfall traps (diameter 8.3 cm, height 10 cm), whose bottom was covered with a sheet of steel mesh (width1 mm), were set twice in the same area for 10 days in July to August 2011. In July 2012,the leaf litter remained wet during the P. japonensisreproductive period because of continuous rainfall, and the main invertebrates that remained in the plot area were individually picked using tweezers.At this time, many S. jasminodora drupes and P. japonensis nymphs were observed on the plot area. A species of Xystodesmidae (Diplopoda), which was often observed near the S. jasminodora fruits in the plot area in 2012, and another species of the burrower bug, Macroscytusjaponensis, were collected from Hinokuma Mountain Park. As a positive control, five P. japonensis nymphs were collected at the same time. In June 2012, 10 Plesiophthalmusnigrocyaneus (Coleoptera) individuals and seven unidentified isopods were collected from the area where P. japonensis nymphs aggregated and fed on S. jasminodora drupes inIsahaya Park, Nagasaki Prefecture. Thirty individual isopods were picked from the leaf litter near the P. japonensis aggregation under three S. jasminodora trees at Satsuma-Sendai, Kagoshima Prefecture (Table 1).

The 46, 87, and 159 invertebrates collected by hand sorting, pitfall traps, and hand picking, respectively, were dissected and soaked in a small volume of tap water in a 6 cm petri dish to release the associated nematodes. Nematodes recovered from the invertebrates were inspected under stereomicroscopes, and thepossible Caenorhabditis species were transferred onto NGM plates seeded with E. coli OP50for further morphological and DNA analyses as described above.

Statistical analysis

Numbers of nematodes on P. japonensis were compared using a Bonferroni/Dunn test (StatView Ver. 4.54, Abacus Concepts, Inc.).

Supplementary Material References

Blaxter ML, De Ley, P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, Vanfleteren JR, Mackey LY, Dorris M, Frisse LM, Vida JT, Thomas WK (1998) A molecular evolutionary framework for the phylum Nematoda. Nature 392:71–75

Iwahori H, Kanzaki N, Futai K (2000) A simple, polymerase chain raction-restriction fragment length polymorphism-aided diagnosis method for pine wilt disease. Forest Pathol 30:157-164

Joyce SA, Reid AP, Driver F, Curran J (1994) Application of polymerase chain reaction (PCR) methods to the identification of entomopathogenic nematodes. In:Burnell AM, Ehlers R-U Masson J-P (eds) COST 812 Biotechnology: Genetics of entomopathogenic nematodes-bacterium complexes. European Commission, Luxembourg, DGXII, 178-187

Sulston J, Hodgkin J (1988) Methods. In:Wood WB, the Community of C. elegans Researchers (eds) The nematode Caenorhabditiselegans. Cold Spring Harbor Laboratory Press,New York, pp 603

Table 1. List of Parastrachiajaponensis collection sites and stages of P. japonensisexamined for detection ofCaenorhabditis japonica.

Place / Stages of P. japonensisexamined / Date
Hinokuma, Saga / All stages*1 / April 2001~ March 2002
Isahaya, Nagasaki / Nymphs / July 2012
Kadogawa, Miyazaki / Nymphs / July 2012
Kinryu, Saga / Nymphs, adults / July 2005
Kochi, Kochi / Adult females / May 2000
Koshi, Kumamoto / Nymphs, adult females / July 2005
Kurume, Fukuoka / Nymphs, adult females / July 2005, September 2012
Kushikino, Kagoshima / Adult females / July 2005
Mikawauchi, Nagasaki / Nymphs, adult females / July 2005, September 2012
Nobeoka, Miyazaki / Nymphs / July 2005
Satsuma-Sendai, Kagoshima / Nymphs / July 2012
Takeo, Saga / Adults*2 / May 2001
Taku, Saga / Adults*2 / May 2001
Tatsugo, Kagoshima / Nymphs / July 2005
Tatsuta, Kumamoto / Nymphs / July 2012

P. japonensis was collected from a single population at each place.

C. japonica was detected on all nymphs and adult females examined but rareon adult males.

*1see Fig. 2 and Fig. 2 in supplementary material for details.

*2C. japonica was detected from P. japonensis females but not from males (see Fig. 1 in supplementary material for details)

Table 2. List of invertebrates collected for detection of Caenorhabditis japonica.

Taxa / Number of individual sacrificed
(Hand sorting from soil sample: September, 2009)
Chilopoda / 10
Diplopoda / 5
Insecta / 7
Coleoptera / 3
Dermaptera / 1
Hemiptera
Macroscytusjaponensis / 3
Isopoda / 24
Venezillo spp. / 11
unknown / 13
Subtotal / 46
(Pitfall trap: July-August, 2011)
Arachnida / 2
Chilopoda / 2
Insecta / 83
Coleoptera / 66
Carabidae / 55
Branchinusscotomedes / 29
Damasterblaptoides / 3
unknown / 26
Staphylinidae / 9
Geotrupidae / 2
Dermaptera / 11
Diptera / 6
Drosophilidae*1 / 6
Subtotal / 87
(Hand pick: July, 2012)
Diplopoda / 34
Xystodesmidae / 34
Insecta / 88
Coleoptera / 11
Plesiophthalmusnigrocyaneus*2 / 10
Scarabaeidae*2 / 1
Hemiptera / 57
Parastrachiajaponensis*4 / 5
Macroscytusjaponensis / 52
Isopoda*3
Sp. 1*2
Sp. 2*3
Sp. 3*3 / 37
7
9
21
Subtotal / 159
Total / 292

*1 Adult flies emerged from fruits of Schoepfiajasminodora.

*2, *3 All invertebrates were collected from Hinokuma Mountain Park, Kanzaki, Saga Prefecture except *2 (Isahaya, Nagasaki Prefecture) and *3 (Satsuma-Sendai, Kagoshima Prefecture).

*4 C. japonica DL was found from only P. japonensis nymphs (average = 18.2 DL/bug; min. = 12; max. = 26).

Fig.1 Female-biased association of C. japonica DL with three different P. japonensis populations.In April,C. japonica were found on the P. japonensis females but not on the males. Each dot indicates the number of DL per insect (n = 10). Only two adult females were available from the Kochi population

Fig. 2 Numbers ofC. japonica DL on P. japonensis during the nymphal period of the insect.

Ten insects of each nymphal stage were used. Bar indicates standard deviations.

1