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Evidence of horizontal transmission of primary and secondary endosymbionts between maize and rice weevils (Sitophilus zeamais and Sitophilus oryzae) and the parasitoid Theocolax elegans
Journal of Stored Products Research 59 (2014) 61e65
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Evidence of horizontal transmission of primary and secondary endosymbionts between maize and rice weevils (Sitophilus zeamais and Sitophilus oryzae) and the parasitoid Theocolax elegans ^a b, c, Luiz O. de Oliveira a, Raul Narciso C. Guedes b, * Gislaine A. Carvalho a, Alberto S. Corre a b c
Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brazil rio, Viçosa, MG 36570-000, Brazil Departamento de Entomologia, Universidade Federal de Viçosa, Av. P.H. Rolfs s/n, Campus Universita ~o Paulo, Piracicaba, SP, Brazil Departamento de Entomologia e Acarologia, ESALQ, Universidade de Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 24 May 2014 Available online
Grain weevils are hosts of two cellular endosymbionts: Wolbachia and “Sitophilus Primary Endosymbiont” (SPE). Wolbachia is a facultative endosymbiont, while SPE is an obligatory endosymbiont. Both Wolbachia and SPE are transmitted vertically, that is, from mother to offspring. There are circumstances in which transmission occurs among conspecific organisms or organisms of distinct species (horizontal transmission), and both vertical and horizontal transmissions play significant roles in shaping the host's ecology and evolution. We found molecular evidence for the horizontal transfer of Wolbachia between the maize weevil (Sitophilus zeamais Motschulsky) and the rice weevil (Sitophilus oryzae (L.)) and evidence of horizontal transfer of Wolbachia and SPE between the maize weevil and the parasitoid Theocolax elegans Westwood (Hymenoptera: Pteromalidae). Using 16S rRNA fragments of both symbionts, we verified the co-existence of two Wolbachia strains in maize weevil individuals from a Mexican population, one of which is typically from this species, while the other is from rice weevils. This finding provides evidence of the horizontal transmission of the endosymbiont between maize and rice weevil and supports the contention of similarity and relatedness between these weevil species. We also observed 100% similarity of 16S rRNA fragments between Wolbachia and SPE sequenced from the weevil parasitoid T. elegans and the maize weevil. This evidence suggests the horizontal transmission of both endosymbionts from the maize weevil to its parasitoid T. elegans. In addition to the importance of these findings for the ecology and evolution of weevils, the potential use of endosymbionts in innovative tactics of arthropod pest management in stored products also deserves attention and remains virtually unexplored. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Symbiosis Mutualism Sitophilus primary endosymbiont Wolbachia
1. Introduction The co-existence of diverse species sharing the same habitat within an environment naturally leads to intricate ecological relationships and shifts in the selective pressure over the organisms. Symbiosis is one such relationship in which stable biological interactions occur between organisms of different species, resulting in an advantage or disadvantage to at least one of the interacting organisms (Majerus, 1999; Moran, 2006). Symbiosis involves a contact interaction prevailing through generations by vertical transmission from the mother to offspring or by horizontal
* Corresponding author. Tel.: þ55 31 3899 4008; fax: þ55 31 3899 4012. E-mail addresses: [email protected], [email protected] (R.N.C. Guedes). http://dx.doi.org/10.1016/j.jspr.2014.05.004 0022-474X/© 2014 Elsevier Ltd. All rights reserved.
transmission between organisms of the same or different species (Werren et al., 2008). Arthropods are common hosts of endosymbiotic bacteria (i.e., living within the body or cells of an organism), which may be primary or secondary (Nardon et al., 2002; Chiel et al., 2009). Primary endosymbionts (or P-endosymbionts) exhibit long-standing obligate associations (>10 million years) with their hosts, usually displaying co-speciation with them (Conord et al., 2008; Moran et al., 2008). In contrast, secondary endosymbionts (or Y-endosymbionts) exhibit relatively recent facultative association with their hosts (Moran, 2006; Moran et al., 2008). Primary endosymbionts are vertically transmitted via the maternal line, while their horizontal transmission is rare (Baumann, 2005; Moran et al., 2008). Secondary endosymbionts may also be vertically transmitted, which is achieved by manipulating host reproduction (Hedges et al., 2008;
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Werren et al., 2008), or horizontally transmitted, leading to occurrence of the endosymbiont in distantly related host species (Russell and Moran, 2005; Gehrer and Vorburger, 2012). The horizontal transmission of endosymbionts is potentially important for the host's ecology and evolution, but the phenomenon is poorly understood, particularly when different species are involved (Werren et al., 1995; Vavre et al., 1999; Huigens et al., 2000; Moran and Dunbar, 2006; Caspi-Fluger et al., 2012). Hostparasitoid associations, for instance, seem to offer opportunities for the horizontal transmission of endosymbionts due to the continuous feeding of the parasitoid larvae on the symbiontcontaminated host (Nieves-Aldrey and Fontal-Cazalla, 1999). Stored-product insects are hosts of primary endosymbionts, and Wolbachia, a secondary endosymbiont, has been detected in different species, though psocids have been the main focus of attention (Mikac, 2007; Dong et al., 2007; Kageyama et al., 2010; Kondo et al., 2011). The growing interest in such interactions, particularly for Wolbachia, is not only due to their relevance in the ecology and evolution of their host but also their potential for arthropod pest management (Bourtzis, 2008; Werren et al., 2008; Brelsfoard and Dobson, 2009; Saridaki and Bourtzis, 2010). Stored-product weevils of the genus Sitophilus are key pests of stored cereals, with ever-increasing problems of insecticide resistance and potential control failures (Perez-Mendoza, 1999; Ribeiro ^a et al., 2011). Sitophilus et al., 2003; Pimentel et al., 2009; Corre weevils exhibit an obligate intracellular endosymbiont, the “Sitophilus Primary Endosymbiont” (SPE), which allegedly provides nutrients and essential vitamins for insect development and is directly involved in insect energy metabolism (Wicker, 1983; Heddi et al., 1999, 2001). These weevils also host the facultative endosymbiont Wolbachia, which is found intra- and extracellularly in the insect hemolymph, allowing for its horizontal transmission (Heddi et al., 1999; Russell and Moran, 2005; Kageyama et al., 2010; Chiel et al., 2009). Here, we present evidence for the horizontal transfer of Wolbachia between the maize and rice weevils (Sitophilus zeamais Motschulsky and Sitophilus oryzae (L.)) and the horizontal transfer of Wolbachia and SPE between the maize weevil and its parasitoid Theocolax elegans Westwood (Hymenoptera: Pteromalidae). 2. Materials and methods 2.1. Population sampling We sampled 11 populations of the maize weevil and 16 populations of the rice weevil from different regions around the world (Table 1). Genitalia inspection (Halstead, 1963) and molecular data ^a et al., 2013) confirmed the identity of the weevil species. (Corre We sampled adults of the parasitoid T. elegans from a maize weevil colony reared in our laboratory; Dr. Marcelo M. Haro (Department of Entomology, Federal University of Lavras, Brazil) confirmed the identity of this parasitoid. The insects were kept in 95% ethanol at 20 C until further use. 2.2. DNA extraction, amplification and sequencing Total genomic DNA was extracted following the method of Clark et al. (2001). Polymerase chain reaction (PCR) amplified a fragment of the 16S ribosomal gene of each endosymbiont using the speciesspecific primers described by Heddi et al. (1999). PCR amplifications were performed in 25 mL containing 3 mL of total DNA (40 ng), 1.0 U of Taq polymerase (Phoneutria, Belo Horizonte, MG, Brazil), 5 buffer (Phoneutria), dNTPs (2.5 mM of each), and 0.4 mM of each primer. The PCR cycles used the following conditions: a 5 min denaturation step at 94 C, followed by 35 cycles of denaturation at
Table 1 Occurrence of primary endosymbionts (SZPE and SOPE) and a secondary endosymbiont (Wolbachia) in populations of the maize weevil (Sitophilus zeamais), rice weevil (S. oryzae), and parasitoid Theocolax elegans. Population
Coordinates
Endosymbionts SZPE SOPE W-SZ W-SO
S. zeamais n, Mexico Tlatizapa La Hormiga, Colombia Ciudad Panama, Panama Lima, Peru Shimla, India Bangkok, Thailand Ipojuca, PE, Brazil Votuporanga, SP, Brazil Maputo, MP, Mozambique Richvale, CA, USA Brisbane, Australia
18 410 0000 N/99 070 0000 W 00 250 0000 N/76 540 0000 W 08 580 4000 N/79 320 2300 W
þ þ þ
þ þ þ
þ
12 31 13 08 20
þ þ þ þ þ
þ þ þ þ þ
þ
þ
39 280 3800 N/121 440 4100 W þ 27 280 2200 S/153 010 4000 E þ
S. oryzae S~ ao Borja, RS, Brazil Cascavel, PR, Brazil cia Kifisia, Gre Lima, Peru Queensland, Austr alia Iran Equador Oklahoma, USA Burma Trinidae & Tobago Shimla, India Egito Georgia, EUA Curitiba, PR, Brazil Lichinga, Maputo Lima, Peru
28 390 3900 S/56 000 1400 W 24 570 2100 S/53 270 1800 W 38 50 000 N/23 490 000 E 12 20 3600 S/77 10 4200 W 23 00 000 S/143 00 000 E 32 00 000 N/53 00 000 E 0 130 700 S/78 300 3500 W 35 280 5600 N/97 320 500 W 19 450 N/96 60 E 10 390 N/61 280 W 31 60 1200 N/77 100 2000 E 30 030 N/31 140 W 31 290 4500 N/83 310 8300 W 25 250 4700 S/49 160 1900 W 13 180 4600 S/35 140 26 E 12 20 3600 S/77 10 4200 W
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
T. elegans Laboratory Strain
20 450 1400 S/42 520 5500 W
þ
þ
020 060 450 230 250
0600 1200 0800 5600 2200
S/77 010 0700 W N/77 100 2000 E N/100 290 3800 E S/35 030 5000 W S/49 580 2200 W
18 35 0000 S/32 350 0000 E
SZPE, Sitophilus zeamais primary endosymbiont; SOPE, Sitophilus oryzae primary endosymbiont; W-SZ, Wolbachia strain from Sitophilus zeamais; W-SO, Wolbachia strain from Sitophilus oryzae.
94 C during 30 s, annealing at 48.7 C (for Wolbachia) or 55.4 C (for both SOPE and SZPE) during 45 s, elongation at 72 C during 90 s, and a final extension step at 72 C during 10 min. PCR amplicons were treated with USB ExoSAP IT (Affymetrix, Santa Barbara, CA, USA) at a ratio of 1.5 mL for each 5 mL. Sequencing was performed by Macrogen Inc., South Korea (www.macrogen.com), using the same primers as the PCR amplifications. All sequences were imported into Sequencher version 4.8 (Gene Codes Corp., Ann Harbor, MI, USA) for editing and sequence alignment. 2.3. Bacterial cloning PCR amplicons obtained from maize weevils collected in n (Mexico) were cloned in the pGEM®T Easy vector Tlatizapa (Promega, Fitchburg, WI, USA). The bacterial clones were subjected to PCR amplification, and the amplicons were purified with the Kit Wizard® Plus Minipreps (Promega) prior to sequencing at Macrogen. 2.4. Analyses of molecular data The sequences obtained as queries in BLAST searches (Altschul et al., 1990) were used against public databases at the National Center for Biotechnology Information. Sequence alignments were used to compare sequences and verify the level of identity between the sequences we obtained and those at public databases.
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3. Results 3.1. Molecular characterization of endosymbionts in weevils Sequencing of the 16S ribosomal gene of either S. zeamais Primary Endosymbiont (SZPE) or S. oryzae Primary Endosymbiont (SOPE) produced a fragment of 530 bases long. There were 32 sequences in the SZPE dataset and 40 sequences in the SOPE dataset. While DNA polymorphisms were absent within each dataset, 12 substitutions distinguished the 16S ribosomal genes of SZPE and SOPE (Fig. 1). SZPE and SOPE were of widespread occurrence; SZPE was confined to maize weevils, whereas SOPE was detected exclusively in rice weevils (Table 1). For the 16S ribosomal gene of Wolbachia, we obtained 38 sequences of 589 bases long. The presence of Wolbachia was detected in nine out of 11 populations of the maize weevil and in seven out of 16 populations of the rice weevil (Table 1). The sequences of Wolbachia were collapsed into two haplotypes (W-SZ and W-SO), and four substitutions set W-SZ apart from W-SO (Fig. 2). W-SZ and WSO occurred exclusively in the maize weevils and rice weevils, n (Mexico), most respectively. With the exception of Tlatizapa populations of weevil presented a single Wolbachia haplotype (see below). The Blast search retrieved several highly similar sequences (Evalues ¼ 0.0) that matched our sequences for SZPE (M85269.1), SOPE (AF005235.1), or Wolbachia (AF035160). The Blast results allowed us to confirm that the PCR amplicons we obtained displayed the expected DNA sequences, thereby confirming their identity. The sequences were deposited in GenBank with the accession numbers KJ854239-KJ854314 (SPE) and KJ854315KJ855361 (Wolbachia).
Fig. 2. Sequence alignment of the variable sites in the 16S ribosomal gene of secondary endosymbionts of Sitophilus zeamais (W-SZ) and S. oryzae (W-SO). The Wolbachia variable sites of the DNA sequence obtained from the weevil parasitoid Theocolax elegans is indicated (W-Te), as is the variable sites from the maize weevil population n (Mexico) (W-Sz/W-So). Alternative occurence of either A and G is from Tlatizapa indicated by R. Each sequence spans 530 bases. The numbers on top indicate the nucleotide position during alignment.
Wolbachia. Sequence alignments indicated 100% sequence identity between the sequences of SZPE from T. elegans and SZPE from maize weevils, as well as Wolbachia (W-SZ) from T. elegans and maize weevil.
3.2. Intraindividual polymorphism for Wolbachia in weevils Inspection of the electropherograms from the 18 specimens of maize weevil from Tlatizap an (Mexico) that we sequenced indicated four overlapping double peaks in the sequences of the 16S ribosomal gene of Wolbachia. One such ambiguity is depicted in Fig. 3. The sequences obtained from bacterially cloned PCR products were aligned with the two haplotypes we recovered for Wolbachia (W-SZ and W-SO) through direct sequencing. Sequence alignments indicated that the clones comprised two distinct sets of sequences, displaying 100% sequence identity with either W-SZ or W-SO. This finding is suggestive of intraindividual polymorphism, which arises when Wolbachia strains carrying polymorphic 16S ribosomal genes co-exist within a single weevil. 3.3. Molecular characterization of endosymbionts in the parasitoid Genomic DNA of all tested individuals of the parasitoid T. elegans yielded positive amplifications for both SZPE and
Fig. 1. Sequence alignment of the variable sites in the 16S ribosomal gene of primary endosymbionts of Sitophilus zeamais (SZPE) and S. oryzae (SOPE). Each sequence spans 530 bases. The numbers on top indicate the nucleotide position during alignment.
Fig. 3. Partial electropherograms from sequences of the 16S ribosomal gene from Wolbachia of maize weevils (Sitophilus zeamais) from population Tlatizapan. Top: Presence of two overlapping double peaks (R ¼ A or G) at position 421. Middle and bottom: sequences from bacterially cloned PCR products displaying no ambiguity.
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4. Discussion Evidence of horizontal transmission of endosymbionts is scarce and is thus believed to rarely occur (Braig et al., 1994; Fujii et al., 2001; Xi et al., 2006). Nonetheless, the lack of congruence between endosymbiont and host phylogenies lends credence to horizontal transmission (Baumann, 2005; Moran et al., 2008). Secondary endosymbionts are more likely to be horizontally transmitted than primary endosymbionts, but in the present study, we provide evidence for the horizontal transmission of both a secondary endosymbiont, Wolbachia, and a primary endosymbiont of the maize weevil, SZPE. The phylogeny of the subfamily Dryophthoridae (Coleoptera: Curculionidae) indicates that maize and rice weevils are sister species. This is also supported by morphological similarities between the two species and their ability to mate, though generating infertile offspring; however, the grain weevil species of the genus Sitophilus still lack a conclusive phylogenetic analysis (Plarre, 2010). Maize and rice weevils also exhibit niche overlap, though the former species is more frequently associated with cereal grains in tropical areas, while the latter is usually associated with temperate and subtropical ^a et al., 2013). conditions (Longstaff, 1981; Rees, 1996; Corre Evidence of horizontal transmission based on the presence of two Wolbachia strains in maize weevil individuals e one of which is typically from this species and the other from rice weevils e provides support for the contention of the similarity and relatedness between these species. This is because they are able to share a common endosymbiont strain recently acquired by both species (as this association is facultative). Furthermore, horizontal transmission between hosts can generate co-infection and consequent opportunity of gene recombination and transfer. Therefore, even if infrequent, horizontal transmission of endosymbionts exhibits potential consequences for the population structure and genome dynamics in these species (Kondo et al., 2005). We observed a high infection frequency of SPE and Wolbachia in the parasitoid T. elegans. The high similarity of the parasitoid endosymbionts with the maize weevil endosymbionts adds evidence to the notion of their horizontal transmission. However, it is still necessary to elucidate the transmission routes within and between species because of their implications to the ecology and evolution of these insects (Chiel et al., 2009). In addition, such knowledge also seems to have the potential for designing alternative management methods for arthropod pest species, including sterile insect techniques (or incompatible insect techniques) and/or insertion of fitness reduction factors leading to pest population suppression or replacement (Werren et al., 2008; Brelsfoard and Dobson, 2009; Saridaki and Bourtzis, 2009, 2010). Within-species horizontal transmission of endosymbionts via coupling and cannibalism has been reported among insects (Huigens et al., 2000; Moran and Dunbar, 2006). Both methods of horizontal transmission may also occur between different, but closely related, species, such as maize and rice weevils. This is because these two weevil species co-occur and are able to mate with one another; they also face an environment subject to intense larval competition, participating in direct interference and cannibalism within the shared grain (Danho et al., 2002; Guedes et al., 2010). Horizontal transmission of endosymbionts between host and parasitoid is likely unidirectional, from the host to the parasitoid, because the host usually dies with the interaction, thereby preventing successful endosymbiont transmission. This is likely the case for Wolbachia and SZPE transmission from the maize weevil to T. elegans. However, the horizontal transmission from T. elegans to the maize weevil may also occur because after the endosymbiont transmission from the parasitoid to immatures of the weevil within the grain, such immatures may be the target of direct interference
during larval competition and subsequent cannibalism, allowing the consequent transmission of the endosymbiont originally from the parasitoid to its host, through the infected immature cannibalistically fed upon. Interspecies horizontal transmission of endosymbionts is rare, and subsequent vertical transmission may be reduced in posterior generations of the infected population (Russell and Moran, 2005; Jaenike et al., 2007; Chiel et al., 2009). These transmissions may however be evolutionarily important if they contribute to endosymbiont fixation in the infected population (Chiel et al., 2009). The likely horizontal transmission of endosymbionts between maize and rice weevils and between the maize weevil and the parasitoid T. elegans is a potential experimental model to elucidate unanswered evolutionary questions pertaining the Sitophilus species and their adaptation to stored cereals. The presence of Wolbachia in maize and rice weevils may have accelerated the speciation process in such case through cytoplasmic incompatibility, which has already been reported in species of Sitophilus (Heddi et al., 1999). The role of parasitoids in such scenarios remains to be assessed, and they may even serve as symbiont vectors among species of this genus. The potential use of endosymbionts in innovative tactics of arthropod pest management in stored products also deserves attention and remains virtually unexplored.
Acknowledgments This contribution was supported by grants from the Minas Gerais State Foundation for Research Aid (FAPEMIG), National Council of Scientific and Technological Development (CNPq), and CAPES Foundation. The technical assistance provided by J. Vieira and L. Pantoja was greatly appreciated, as was the assistance of the different colleagues who sent us specimens of weevil populations. We also appreciated the assistance of Dr. M. Haro in identifying the weevil parasitoid species.
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Evidence of horizontal transmission of primary and secondary endosymbionts between maize and rice weevils (Sitophilus zeamais and Sitophilus oryzae) and the parasitoid Theocolax elegans ^a b, c, Luiz O. de Oliveira a, Raul Narciso C. Guedes b, * Gislaine A. Carvalho a, Alberto S. Corre a b c
Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brazil rio, Viçosa, MG 36570-000, Brazil Departamento de Entomologia, Universidade Federal de Viçosa, Av. P.H. Rolfs s/n, Campus Universita ~o Paulo, Piracicaba, SP, Brazil Departamento de Entomologia e Acarologia, ESALQ, Universidade de Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 24 May 2014 Available online
Grain weevils are hosts of two cellular endosymbionts: Wolbachia and “Sitophilus Primary Endosymbiont” (SPE). Wolbachia is a facultative endosymbiont, while SPE is an obligatory endosymbiont. Both Wolbachia and SPE are transmitted vertically, that is, from mother to offspring. There are circumstances in which transmission occurs among conspecific organisms or organisms of distinct species (horizontal transmission), and both vertical and horizontal transmissions play significant roles in shaping the host's ecology and evolution. We found molecular evidence for the horizontal transfer of Wolbachia between the maize weevil (Sitophilus zeamais Motschulsky) and the rice weevil (Sitophilus oryzae (L.)) and evidence of horizontal transfer of Wolbachia and SPE between the maize weevil and the parasitoid Theocolax elegans Westwood (Hymenoptera: Pteromalidae). Using 16S rRNA fragments of both symbionts, we verified the co-existence of two Wolbachia strains in maize weevil individuals from a Mexican population, one of which is typically from this species, while the other is from rice weevils. This finding provides evidence of the horizontal transmission of the endosymbiont between maize and rice weevil and supports the contention of similarity and relatedness between these weevil species. We also observed 100% similarity of 16S rRNA fragments between Wolbachia and SPE sequenced from the weevil parasitoid T. elegans and the maize weevil. This evidence suggests the horizontal transmission of both endosymbionts from the maize weevil to its parasitoid T. elegans. In addition to the importance of these findings for the ecology and evolution of weevils, the potential use of endosymbionts in innovative tactics of arthropod pest management in stored products also deserves attention and remains virtually unexplored. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Symbiosis Mutualism Sitophilus primary endosymbiont Wolbachia
1. Introduction The co-existence of diverse species sharing the same habitat within an environment naturally leads to intricate ecological relationships and shifts in the selective pressure over the organisms. Symbiosis is one such relationship in which stable biological interactions occur between organisms of different species, resulting in an advantage or disadvantage to at least one of the interacting organisms (Majerus, 1999; Moran, 2006). Symbiosis involves a contact interaction prevailing through generations by vertical transmission from the mother to offspring or by horizontal
* Corresponding author. Tel.: þ55 31 3899 4008; fax: þ55 31 3899 4012. E-mail addresses: [email protected], [email protected] (R.N.C. Guedes). http://dx.doi.org/10.1016/j.jspr.2014.05.004 0022-474X/© 2014 Elsevier Ltd. All rights reserved.
transmission between organisms of the same or different species (Werren et al., 2008). Arthropods are common hosts of endosymbiotic bacteria (i.e., living within the body or cells of an organism), which may be primary or secondary (Nardon et al., 2002; Chiel et al., 2009). Primary endosymbionts (or P-endosymbionts) exhibit long-standing obligate associations (>10 million years) with their hosts, usually displaying co-speciation with them (Conord et al., 2008; Moran et al., 2008). In contrast, secondary endosymbionts (or Y-endosymbionts) exhibit relatively recent facultative association with their hosts (Moran, 2006; Moran et al., 2008). Primary endosymbionts are vertically transmitted via the maternal line, while their horizontal transmission is rare (Baumann, 2005; Moran et al., 2008). Secondary endosymbionts may also be vertically transmitted, which is achieved by manipulating host reproduction (Hedges et al., 2008;
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Werren et al., 2008), or horizontally transmitted, leading to occurrence of the endosymbiont in distantly related host species (Russell and Moran, 2005; Gehrer and Vorburger, 2012). The horizontal transmission of endosymbionts is potentially important for the host's ecology and evolution, but the phenomenon is poorly understood, particularly when different species are involved (Werren et al., 1995; Vavre et al., 1999; Huigens et al., 2000; Moran and Dunbar, 2006; Caspi-Fluger et al., 2012). Hostparasitoid associations, for instance, seem to offer opportunities for the horizontal transmission of endosymbionts due to the continuous feeding of the parasitoid larvae on the symbiontcontaminated host (Nieves-Aldrey and Fontal-Cazalla, 1999). Stored-product insects are hosts of primary endosymbionts, and Wolbachia, a secondary endosymbiont, has been detected in different species, though psocids have been the main focus of attention (Mikac, 2007; Dong et al., 2007; Kageyama et al., 2010; Kondo et al., 2011). The growing interest in such interactions, particularly for Wolbachia, is not only due to their relevance in the ecology and evolution of their host but also their potential for arthropod pest management (Bourtzis, 2008; Werren et al., 2008; Brelsfoard and Dobson, 2009; Saridaki and Bourtzis, 2010). Stored-product weevils of the genus Sitophilus are key pests of stored cereals, with ever-increasing problems of insecticide resistance and potential control failures (Perez-Mendoza, 1999; Ribeiro ^a et al., 2011). Sitophilus et al., 2003; Pimentel et al., 2009; Corre weevils exhibit an obligate intracellular endosymbiont, the “Sitophilus Primary Endosymbiont” (SPE), which allegedly provides nutrients and essential vitamins for insect development and is directly involved in insect energy metabolism (Wicker, 1983; Heddi et al., 1999, 2001). These weevils also host the facultative endosymbiont Wolbachia, which is found intra- and extracellularly in the insect hemolymph, allowing for its horizontal transmission (Heddi et al., 1999; Russell and Moran, 2005; Kageyama et al., 2010; Chiel et al., 2009). Here, we present evidence for the horizontal transfer of Wolbachia between the maize and rice weevils (Sitophilus zeamais Motschulsky and Sitophilus oryzae (L.)) and the horizontal transfer of Wolbachia and SPE between the maize weevil and its parasitoid Theocolax elegans Westwood (Hymenoptera: Pteromalidae). 2. Materials and methods 2.1. Population sampling We sampled 11 populations of the maize weevil and 16 populations of the rice weevil from different regions around the world (Table 1). Genitalia inspection (Halstead, 1963) and molecular data ^a et al., 2013) confirmed the identity of the weevil species. (Corre We sampled adults of the parasitoid T. elegans from a maize weevil colony reared in our laboratory; Dr. Marcelo M. Haro (Department of Entomology, Federal University of Lavras, Brazil) confirmed the identity of this parasitoid. The insects were kept in 95% ethanol at 20 C until further use. 2.2. DNA extraction, amplification and sequencing Total genomic DNA was extracted following the method of Clark et al. (2001). Polymerase chain reaction (PCR) amplified a fragment of the 16S ribosomal gene of each endosymbiont using the speciesspecific primers described by Heddi et al. (1999). PCR amplifications were performed in 25 mL containing 3 mL of total DNA (40 ng), 1.0 U of Taq polymerase (Phoneutria, Belo Horizonte, MG, Brazil), 5 buffer (Phoneutria), dNTPs (2.5 mM of each), and 0.4 mM of each primer. The PCR cycles used the following conditions: a 5 min denaturation step at 94 C, followed by 35 cycles of denaturation at
Table 1 Occurrence of primary endosymbionts (SZPE and SOPE) and a secondary endosymbiont (Wolbachia) in populations of the maize weevil (Sitophilus zeamais), rice weevil (S. oryzae), and parasitoid Theocolax elegans. Population
Coordinates
Endosymbionts SZPE SOPE W-SZ W-SO
S. zeamais n, Mexico Tlatizapa La Hormiga, Colombia Ciudad Panama, Panama Lima, Peru Shimla, India Bangkok, Thailand Ipojuca, PE, Brazil Votuporanga, SP, Brazil Maputo, MP, Mozambique Richvale, CA, USA Brisbane, Australia
18 410 0000 N/99 070 0000 W 00 250 0000 N/76 540 0000 W 08 580 4000 N/79 320 2300 W
þ þ þ
þ þ þ
þ
12 31 13 08 20
þ þ þ þ þ
þ þ þ þ þ
þ
þ
39 280 3800 N/121 440 4100 W þ 27 280 2200 S/153 010 4000 E þ
S. oryzae S~ ao Borja, RS, Brazil Cascavel, PR, Brazil cia Kifisia, Gre Lima, Peru Queensland, Austr alia Iran Equador Oklahoma, USA Burma Trinidae & Tobago Shimla, India Egito Georgia, EUA Curitiba, PR, Brazil Lichinga, Maputo Lima, Peru
28 390 3900 S/56 000 1400 W 24 570 2100 S/53 270 1800 W 38 50 000 N/23 490 000 E 12 20 3600 S/77 10 4200 W 23 00 000 S/143 00 000 E 32 00 000 N/53 00 000 E 0 130 700 S/78 300 3500 W 35 280 5600 N/97 320 500 W 19 450 N/96 60 E 10 390 N/61 280 W 31 60 1200 N/77 100 2000 E 30 030 N/31 140 W 31 290 4500 N/83 310 8300 W 25 250 4700 S/49 160 1900 W 13 180 4600 S/35 140 26 E 12 20 3600 S/77 10 4200 W
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
T. elegans Laboratory Strain
20 450 1400 S/42 520 5500 W
þ
þ
020 060 450 230 250
0600 1200 0800 5600 2200
S/77 010 0700 W N/77 100 2000 E N/100 290 3800 E S/35 030 5000 W S/49 580 2200 W
18 35 0000 S/32 350 0000 E
SZPE, Sitophilus zeamais primary endosymbiont; SOPE, Sitophilus oryzae primary endosymbiont; W-SZ, Wolbachia strain from Sitophilus zeamais; W-SO, Wolbachia strain from Sitophilus oryzae.
94 C during 30 s, annealing at 48.7 C (for Wolbachia) or 55.4 C (for both SOPE and SZPE) during 45 s, elongation at 72 C during 90 s, and a final extension step at 72 C during 10 min. PCR amplicons were treated with USB ExoSAP IT (Affymetrix, Santa Barbara, CA, USA) at a ratio of 1.5 mL for each 5 mL. Sequencing was performed by Macrogen Inc., South Korea (www.macrogen.com), using the same primers as the PCR amplifications. All sequences were imported into Sequencher version 4.8 (Gene Codes Corp., Ann Harbor, MI, USA) for editing and sequence alignment. 2.3. Bacterial cloning PCR amplicons obtained from maize weevils collected in n (Mexico) were cloned in the pGEM®T Easy vector Tlatizapa (Promega, Fitchburg, WI, USA). The bacterial clones were subjected to PCR amplification, and the amplicons were purified with the Kit Wizard® Plus Minipreps (Promega) prior to sequencing at Macrogen. 2.4. Analyses of molecular data The sequences obtained as queries in BLAST searches (Altschul et al., 1990) were used against public databases at the National Center for Biotechnology Information. Sequence alignments were used to compare sequences and verify the level of identity between the sequences we obtained and those at public databases.
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3. Results 3.1. Molecular characterization of endosymbionts in weevils Sequencing of the 16S ribosomal gene of either S. zeamais Primary Endosymbiont (SZPE) or S. oryzae Primary Endosymbiont (SOPE) produced a fragment of 530 bases long. There were 32 sequences in the SZPE dataset and 40 sequences in the SOPE dataset. While DNA polymorphisms were absent within each dataset, 12 substitutions distinguished the 16S ribosomal genes of SZPE and SOPE (Fig. 1). SZPE and SOPE were of widespread occurrence; SZPE was confined to maize weevils, whereas SOPE was detected exclusively in rice weevils (Table 1). For the 16S ribosomal gene of Wolbachia, we obtained 38 sequences of 589 bases long. The presence of Wolbachia was detected in nine out of 11 populations of the maize weevil and in seven out of 16 populations of the rice weevil (Table 1). The sequences of Wolbachia were collapsed into two haplotypes (W-SZ and W-SO), and four substitutions set W-SZ apart from W-SO (Fig. 2). W-SZ and WSO occurred exclusively in the maize weevils and rice weevils, n (Mexico), most respectively. With the exception of Tlatizapa populations of weevil presented a single Wolbachia haplotype (see below). The Blast search retrieved several highly similar sequences (Evalues ¼ 0.0) that matched our sequences for SZPE (M85269.1), SOPE (AF005235.1), or Wolbachia (AF035160). The Blast results allowed us to confirm that the PCR amplicons we obtained displayed the expected DNA sequences, thereby confirming their identity. The sequences were deposited in GenBank with the accession numbers KJ854239-KJ854314 (SPE) and KJ854315KJ855361 (Wolbachia).
Fig. 2. Sequence alignment of the variable sites in the 16S ribosomal gene of secondary endosymbionts of Sitophilus zeamais (W-SZ) and S. oryzae (W-SO). The Wolbachia variable sites of the DNA sequence obtained from the weevil parasitoid Theocolax elegans is indicated (W-Te), as is the variable sites from the maize weevil population n (Mexico) (W-Sz/W-So). Alternative occurence of either A and G is from Tlatizapa indicated by R. Each sequence spans 530 bases. The numbers on top indicate the nucleotide position during alignment.
Wolbachia. Sequence alignments indicated 100% sequence identity between the sequences of SZPE from T. elegans and SZPE from maize weevils, as well as Wolbachia (W-SZ) from T. elegans and maize weevil.
3.2. Intraindividual polymorphism for Wolbachia in weevils Inspection of the electropherograms from the 18 specimens of maize weevil from Tlatizap an (Mexico) that we sequenced indicated four overlapping double peaks in the sequences of the 16S ribosomal gene of Wolbachia. One such ambiguity is depicted in Fig. 3. The sequences obtained from bacterially cloned PCR products were aligned with the two haplotypes we recovered for Wolbachia (W-SZ and W-SO) through direct sequencing. Sequence alignments indicated that the clones comprised two distinct sets of sequences, displaying 100% sequence identity with either W-SZ or W-SO. This finding is suggestive of intraindividual polymorphism, which arises when Wolbachia strains carrying polymorphic 16S ribosomal genes co-exist within a single weevil. 3.3. Molecular characterization of endosymbionts in the parasitoid Genomic DNA of all tested individuals of the parasitoid T. elegans yielded positive amplifications for both SZPE and
Fig. 1. Sequence alignment of the variable sites in the 16S ribosomal gene of primary endosymbionts of Sitophilus zeamais (SZPE) and S. oryzae (SOPE). Each sequence spans 530 bases. The numbers on top indicate the nucleotide position during alignment.
Fig. 3. Partial electropherograms from sequences of the 16S ribosomal gene from Wolbachia of maize weevils (Sitophilus zeamais) from population Tlatizapan. Top: Presence of two overlapping double peaks (R ¼ A or G) at position 421. Middle and bottom: sequences from bacterially cloned PCR products displaying no ambiguity.
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4. Discussion Evidence of horizontal transmission of endosymbionts is scarce and is thus believed to rarely occur (Braig et al., 1994; Fujii et al., 2001; Xi et al., 2006). Nonetheless, the lack of congruence between endosymbiont and host phylogenies lends credence to horizontal transmission (Baumann, 2005; Moran et al., 2008). Secondary endosymbionts are more likely to be horizontally transmitted than primary endosymbionts, but in the present study, we provide evidence for the horizontal transmission of both a secondary endosymbiont, Wolbachia, and a primary endosymbiont of the maize weevil, SZPE. The phylogeny of the subfamily Dryophthoridae (Coleoptera: Curculionidae) indicates that maize and rice weevils are sister species. This is also supported by morphological similarities between the two species and their ability to mate, though generating infertile offspring; however, the grain weevil species of the genus Sitophilus still lack a conclusive phylogenetic analysis (Plarre, 2010). Maize and rice weevils also exhibit niche overlap, though the former species is more frequently associated with cereal grains in tropical areas, while the latter is usually associated with temperate and subtropical ^a et al., 2013). conditions (Longstaff, 1981; Rees, 1996; Corre Evidence of horizontal transmission based on the presence of two Wolbachia strains in maize weevil individuals e one of which is typically from this species and the other from rice weevils e provides support for the contention of the similarity and relatedness between these species. This is because they are able to share a common endosymbiont strain recently acquired by both species (as this association is facultative). Furthermore, horizontal transmission between hosts can generate co-infection and consequent opportunity of gene recombination and transfer. Therefore, even if infrequent, horizontal transmission of endosymbionts exhibits potential consequences for the population structure and genome dynamics in these species (Kondo et al., 2005). We observed a high infection frequency of SPE and Wolbachia in the parasitoid T. elegans. The high similarity of the parasitoid endosymbionts with the maize weevil endosymbionts adds evidence to the notion of their horizontal transmission. However, it is still necessary to elucidate the transmission routes within and between species because of their implications to the ecology and evolution of these insects (Chiel et al., 2009). In addition, such knowledge also seems to have the potential for designing alternative management methods for arthropod pest species, including sterile insect techniques (or incompatible insect techniques) and/or insertion of fitness reduction factors leading to pest population suppression or replacement (Werren et al., 2008; Brelsfoard and Dobson, 2009; Saridaki and Bourtzis, 2009, 2010). Within-species horizontal transmission of endosymbionts via coupling and cannibalism has been reported among insects (Huigens et al., 2000; Moran and Dunbar, 2006). Both methods of horizontal transmission may also occur between different, but closely related, species, such as maize and rice weevils. This is because these two weevil species co-occur and are able to mate with one another; they also face an environment subject to intense larval competition, participating in direct interference and cannibalism within the shared grain (Danho et al., 2002; Guedes et al., 2010). Horizontal transmission of endosymbionts between host and parasitoid is likely unidirectional, from the host to the parasitoid, because the host usually dies with the interaction, thereby preventing successful endosymbiont transmission. This is likely the case for Wolbachia and SZPE transmission from the maize weevil to T. elegans. However, the horizontal transmission from T. elegans to the maize weevil may also occur because after the endosymbiont transmission from the parasitoid to immatures of the weevil within the grain, such immatures may be the target of direct interference
during larval competition and subsequent cannibalism, allowing the consequent transmission of the endosymbiont originally from the parasitoid to its host, through the infected immature cannibalistically fed upon. Interspecies horizontal transmission of endosymbionts is rare, and subsequent vertical transmission may be reduced in posterior generations of the infected population (Russell and Moran, 2005; Jaenike et al., 2007; Chiel et al., 2009). These transmissions may however be evolutionarily important if they contribute to endosymbiont fixation in the infected population (Chiel et al., 2009). The likely horizontal transmission of endosymbionts between maize and rice weevils and between the maize weevil and the parasitoid T. elegans is a potential experimental model to elucidate unanswered evolutionary questions pertaining the Sitophilus species and their adaptation to stored cereals. The presence of Wolbachia in maize and rice weevils may have accelerated the speciation process in such case through cytoplasmic incompatibility, which has already been reported in species of Sitophilus (Heddi et al., 1999). The role of parasitoids in such scenarios remains to be assessed, and they may even serve as symbiont vectors among species of this genus. The potential use of endosymbionts in innovative tactics of arthropod pest management in stored products also deserves attention and remains virtually unexplored.
Acknowledgments This contribution was supported by grants from the Minas Gerais State Foundation for Research Aid (FAPEMIG), National Council of Scientific and Technological Development (CNPq), and CAPES Foundation. The technical assistance provided by J. Vieira and L. Pantoja was greatly appreciated, as was the assistance of the different colleagues who sent us specimens of weevil populations. We also appreciated the assistance of Dr. M. Haro in identifying the weevil parasitoid species.
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