Laboratory of Applied Zoology, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki, Japan;
Department of Entomology, University of Arizona;
Laboratory of Apiculture, Department of Animal Genetics, National Institute of Animal Industry, Tsukuba, Ibaraki, Japan
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Abstract |
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Introduction |
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The occurrence of horizontal transfer of MLEs is generally accepted (Hartl et al. 1997
), however, there are many unanswered questions in MLE evolution. For example, the mechanisms of horizontal transfer remain unknown. Because the intimacy of parasitism may facilitate horizontal transfer (Kidwell 1992a
), we have started to survey MLEs in parasitoid insects and their hosts. Here, we show that a parasitoid wasp and its moth host carry MLEs with 97.6% identity in nucleotide sequence. This high similarity and the lack of MLEs in a congeneric wasp suggest that MLEs were recently transferred horizontally from one of the species to the other one, most likely from the host to the parasitoid.
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Materials and Methods |
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PCR Amplification
Genomic DNA was extracted from adults of the parasitic wasp and pupae of the moth following methods modified from those used for Drosophila (Daniels and Strausbaugh 1986
). The oligonucleotide primers used in this study are shown in table 1
. The degenerate MAR 124F and MAR 276R primers were designed to amplify MLEs from insects, based on the conserved region of mariner transposases (Robertson 1993
). The other primers were used to extend analyses of the PCR products amplified by the MAR 124F-MAR 276R primers (see below). PCR was performed under the following conditions. Template DNA (200 ng) was added to PCR buffer (Boehringer Mannheim) containing 200 µM of each dNTP, 2 mM oligonucleotide primers, and 0.5 U Taq DNA polymerase (Boehringer Mannheim) in 100 µl (total volume). Temperature cycling was carried out in a thermal cycler (Gene Amp 9600, Perkin Elmer) for 30 cycles (1 min at 95°C, 1 min at 55°C, and 1 min at 72°C).
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Isolation of Full-Length MLEs
Genomic libraries of both species were constructed using pUC 19 plasmids digested with PstI and EcoRI. Methods for probe labeling, hybridization, and signal detection are described below in the Southern Blot Analysis section. The nucleotide sequences of positive genomic clones were determined by primer walking using synthetic oligonucleotides.
Computer Analysis of the Sequences
The sequence alignment was performed using GENETYX-MAC, version 10.1 (Software Development). A similarity search for nucleotide and amino acid sequences was carried out with BLAST (Altschul et al. 1990
) using the GenBank and EMBL databases. Phylogenetic reconstruction was performed by PAUP, version 4.0b6 (Sinauer), using maximum parsimony; bootstrap values for the nodes were determined using 100 replicates.
Southern Blot Analysis
The ProbeF and ProbeR primers, internal to the degenerate MAR 124F and MAR 276R primers, were designed from the sequences of the MLEs of the wasp and the moth (fig. 1
). PCR fragments, amplified from moth genomic DNA using the ProbeF and ProbeR primers (table 1
), were cloned into the pCR vector. One clone, designated pFR1, was labeled with digoxigenin-11-dUTP using the PCR DIG Labeling Kit (Boehringer Mannheim). Genomic DNA (10 µg) was digested with EcoRI, and DNA fragments were separated by electrophoresis in a 1% agarose gel and transferred to nylon membranes (Boehringer Mannheim) by capillary blotting. The membranes were prehybridized at 65°C for 4 h in 5 x SSC containing 1% blocking reagent (Boehringer Mannheim), 0.02% SDS, and 0.1% N-lauroylsarcosine. The prehybridization buffer was replaced with fresh buffer containing heat-denatured probe. Hybridization was performed at 65°C for approximately 16 h. Membranes were washed twice with 2 x SSC containing 0.1% SDS for 5 min at room temperature and twice with 0.1% SSC containing 0.1% SDS for 15 min at 65°C. Positive signals were detected using a DIG DNA Detection Kit following the methods recommended by the manufacturer (Boehringer Mannheim).
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Results and Discussion |
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We extracted full-length genomic copies of these MLEs to examine them further. EcoRI/PstI-digested pUC19 genomic DNA libraries of the two species were screened with DIG-labeled PCR products and cloned into the pCR plasmid vector (Invitrogen) (fig. 1a ). From the library for A. reticulatus, three complete genomic copies (named W1, W5, and W6) were isolated (fig. 2a ). One of these elements, W1, carried a 57-bp duplication in the middle of the element, but these three copies were otherwise almost identical. Two elements (named M1 and M4) were isolated from the A. honmai library (fig. 2a ). M1 and M4 were identical, except that M1 had an imperfect duplication of 7 bp and was truncated at the 3' end of the element. (The 3' deletion of M1 was after position 1105. We sequenced 890 bp further without finding more mariner-related sequence.) All of the clones had 25-bp inverted terminal repeats (ITRs) and a TA duplication at the target insertion sites (except for the 5' end of W6, which had TT, and possibly the missing 3' end of M1). These features are common to all Tc1/mariner family transposable elements. These MLEs all possessed stop codons within the putative open reading frame (ORF); therefore, they seem to be inactive. A phylogenetic tree demonstrates the relatedness of these MLE sequences to each other and to MLEs from other species obtained from GenBank by BLAST searching for the most similar sequences in the databases (fig. 3 ). The tree shown was constructed using maximum parsimony, but trees constructed using maximum likelihood and the unweighted pair grouping method with arithmetic means produced the same topology.
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Several issues need to be addressed. The first question is that of what kind of intermediates are involved in horizontal transfer. Intermediate vectors are probably required for the horizontal transfer because transposable elements are not capable of moving from one species to another directly. There are reports of viruses carrying transposable elements (e.g., Miller and Miller 1982
), and viruses might therefore act as shuttles of transposable elements for horizontal transfer (Fraser 1986
). It is known that viruses carried by parasitoids can play an important role in suppression of the host's immune response, thereby preventing encapsulation of the parasitoid egg (Strand and Pech 1995
). It is possible that viruses are (or were) present in the Ascogaster-Adoxophyes system and therefore could act (or could have acted) as vectors to carry transposable elements between the species.
The second question to be addressed is that of the direction of horizontal transfer. We performed Southern blot analysis (fig. 6a
) and PCR assays (fig. 6b
) on two other sibling species of Adoxophyes moths and another species of the wasp genus Ascogaster. MLEs were detected in the summer fruit tortrix Adoxophyes orana fasciata (Yoshiyama et al. 2000
) and the tea tortrix Homona magnanima, but no MLEs were detected in the parasitoid wasp Ascogaster quadridentata. This suggests that transfer was from the host to the parasitoid. This direction is more reasonable than the reverse for another reason: infection of a host by a parasitoid usually leads to the death of the host, thereby precluding successful transfer from the parasitoid to the host. We conclude that the pathway of horizontal transfer was probably from the moth to the wasp.
If this scenario is true, we can infer the sequence of the MLE at the time of transfer. Our analysis of the consensus sequence of the wasp MLEs, along with the comparison with the moth-derived M4 and the lacewing sequences, allows us to infer that at the time of horizontal transfer, the MLE that was transferred must have had the amino acid sequence of the wasp consensus sequence.
The third question to be addressed is whether or not horizontal transfer is common in host-parasite systems. There are few relevant data. P elements of Drosophila are thought to have been introduced into Drosophila melanogaster by a horizontal transfer from Drosophila willistoni (Daniels et al. 1990
; Kidwell 1992b
). That transfer has been postulated to have been effected by physical transfer of P elements from an egg of one species to an egg of the other by the egg predatory mite Proctolaelaps regalis (Houck et al. 1991
). In this case, the P element is not present in the genome of the mite, and the ecological relationship between the mite and the Drosophila species is not particularly close. To our knowledge, there are no other reports suggesting that an especially intimate ecological proximity, such as a host-parasitoid or host-parasite relationship, contributed to horizontal transfer of a transposable element.
In an effort to extend our results, we tested four other pairs of hosts and parasitoids for MLEs (table 2 ). Among these, only one host species (Mythimna separata) and no parasitoids had MLEs. The attractive hypothesis that the special intimacy of the host-parasitoid relationship could facilitate horizontal transfer of transposable elements is thus far demonstrated only in our single example.
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Conclusions |
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Acknowledgements |
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Footnotes |
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1 Present address: USDA-ARS, Department of Entomology, Purdue University.
2 Present address: Department of Biochemistry, Virginia Tech.
3 Abbreviations: ITR, inverted terminal repeat; MLE, mariner-like element.
4 Keywords: mariner-like elements
horizontal transfer
parasitoid
5 Address for correspondence and reprints: Kiyoshi Kimura, Laboratory of Apiculture, Department of Animal Genetics, National Institute of Animal Industry, Tsukuba Norin Danchi P.O. Box 5, Tsukuba, Ibaraki 305-0901, Japan. kimura{at}niai.affrc.go.jp
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References |
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Altschul S. F., W. Gish, W. Miller, E. W. Myers, D. J. Lipman, 1990 Basic local alignment search tool J. Mol. Biol 251:403-410
Daniels S. B., K. R. Peterson, L. D. Strausbaugh, M. G. Kidwell, A. Chovnick, 1990 Evidence for horizontal transmission of the P transposable element between Drosophila species Genetics 124:339-355
Daniels S. B., L. D. Strausbaugh, 1986 The distribution of P element sequences in Drosophila: the willistoni and saltans species groups J. Mol. Evol 23:138-148[ISI][Medline]
Fraser M. J., 1986 Transposon-mediated mutagenesis of baculoviruses: transposon shuttling and implications for speciation Ann. Entomol. Soc. Am 79:773-783[ISI]
Hartl D. L., E. R. Lozovskaya, D. I. Nurminsky, A. R. Lohe, 1997 What restricts the activity of mariner-like transposable elements? Trends Genet 13:197-201[ISI][Medline]
Honda T., Y. Kainoh, H. Honda, 1999 Persistence of learned response in the egg-larval parasitoid, Ascogaster reticulatus Watanabe (Hymenoptera: Braconidae) Entomol. Sci 2:335-340
Houck M. A., J. B. Clark, K. R. Peterson, M. G. Kidwell, 1991 Possible horizontal transfer of Drosophila genes by the mite Proctolaelaps regalis Science 253:1125-1129[ISI][Medline]
Jacobson J. W., M. M. Medhora, D. L. Hartl, 1986 Molecular structure of a somatically unstable transposable element in Drosophila Proc. Natl. Acad. Sci. USA 83:8684-8688[Abstract]
Kidwell M. G., 1992a Horizontal transfer Curr. Opin. Genet. Dev 2:868-873[Medline]
. 1992b Horizontal transfer of P elements and other short inverted repeat transposons Genetica 86:275-286[ISI][Medline]
Maruyama K., D. L. Hartl, 1991 Evidence for interspecific transfer of the transposable element mariner between Drosophila and Zaprionus J. Mol. Evol 33:514-524[ISI][Medline]
Miller D. W., L. K. Miller, 1982 A virus mutant with an insertion of a copia-like transposable element Nature 299:562-564[ISI][Medline]
Noguchi H., 1993 Smaller tea tortrix, Summer fruit tortrix and Oriental tea tortrix Pp. 9196 in K. Yushima, S. Kamano, and Y. Tamaki, eds. Rearing methods in insects. Japan Plant Protection Association, Tokyo [in Japanese]
Robertson H. M., 1993 The mariner transposable element is widespread in insects Nature 362:241-245[ISI][Medline]
. 1995 Tc1-mariner superfamily of transposons in animals J. Insect Physiol 41:99-105[ISI]
Robertson H. M., D. J. Lampe, 1995a Distribution of transposable elements in arthropods Annu. Rev. Entomol 40:333-357[ISI][Medline]
. 1995b Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera Mol. Biol. Evol 12:850-862[Abstract]
Robertson H. M., D. J. Lampe, E. G. MacLeod, 1992 A mariner transposable element from a lacewing Nucleic Acids Res 20:6409[ISI][Medline]
Robertson H. M., E. G. MacLeod, 1993 Five major subfamilies of mariner transposable elements in insects, including the Mediterranean fruit fly, and related arthropods Insect Mol. Biol 2:125-139[Medline]
Strand M. R., L. L. Pech, 1995 Immunological basis for compatibility in parasitoid-host relationships Annu. Rev. Entomol 40:31-56[ISI][Medline]
Syvanen M., 1994 Horizontal gene transfer: evidence and possible consequences Annu. Rev. Genet 28:237-261[ISI][Medline]
Watanabe C., 1967 Description of a new species of the genus Ascogaster Wesmael and notes on synonymy of Apanteles species Insecta Matsumurana 29:41-44
Yoshiyama M., H. Honda, H. Noguchi, K. Kimura, 2000 Analysis of mariner-like elements in the smaller tea tortrix, Adoxophyes honmai and the summer fruit tortrix, Adoxophyes orana fasciata (Lepidoptera: Tortricidae) Appl. Entomol. Zool 35:313-320[ISI]