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Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil
G Model TTBDIS-807; No. of Pages 3
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil Marcos Antônio Bezerra Santos a , Lucia Oliveira de Macedo a , Islanne Barbosa de Souza a , Carlos Alberto do Nascimento Ramos b , Leucio Câmara Alves c , Rafael Antonio Nascimento Ramos a,∗ , Gílcia Aparecida de Carvalho a a
Unidade Acadêmica de Garanhuns, Universidade Federal Rural de Pernambuco, Garanhuns, Brazil Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Campo Grande, Brazil c Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, Brazil b
a r t i c l e
i n f o
Article history: Received 10 January 2017 Received in revised form 12 March 2017 Accepted 15 March 2017 Available online xxx Keywords: Ticks Parasitoid Biological control Ixodiphagus hookeri
a b s t r a c t The biological control of ticks represents an alternative method to the chemical control, given its ecological-friendly approach. Amongst the alternatives, the use of parasitoids of the genus Ixodiphagus (Hymenoptera: Encyrtidae) has been largely investigated. The aim of this study was to document and molecularly characterize Ixodiphagus wasps in ticks from a tropical region of Brazil. From October 2015 to March 2016, Rhipicephalus sanguineus sensu lato ticks (n = 1814) were collected from naturally infested dogs and Ixodiphagus larvae were detected by microscopic examination. In addition, adult wasps were obtained in the laboratory. Larvae and adults were molecularly identified as Ixodiphagus hookeri. These findings suggest that this type of parasitism deserves to be studied in local tick populations, in order to elucidate the role of these wasps as a potential alternative to chemical tick control. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Ticks are amongst the most important vectors of a wide range of pathogens, including protozoa, rickettsiae, spirochetes, viruses and nematodes (Ramos et al., 2015; Hubálek and Rudolf, 2012). In addition, these ectoparasites may cause irritation, allergies, intoxication, and paralysis of their hosts. The control of these arthropods is of great importance for medical and veterinary science, given their global economic and public health impact. Over the last few years, the biological control of ticks using predators, parasitoids and arthropod pathogens has been investigated as an alternative to chemical control. Amongst these tools, parasitoids of the genus Ixodiphagus (Hymenoptera: Encyrtidae) have been documented in different tick species, such as Amblyomma spp. (Mwangi et al., 1997; Lopes et al., 2012), Ixodes spp. (Heath and Cane, 2010; Ramos et al., 2015), Dermacentor spp. (Wood, 1911), Haemaphysalis spp. (Doube and Heath, 1975) and Rhipicephalus sanguineus sensu lato (s.l.) (Coronado, 2006; Lopes et al., 2012). The efficacy of larvae of the genus Ixodiphagus in tick control is still debated among researchers since the biology of
∗ Corresponding author. E-mail address: [email protected] (R.A.N. Ramos).
these wasps has not yet been completely elucidated (Collatz et al., 2011). The most important study conducted so far, demonstrated the ability of this parasitoid to reduce the number of Amblyomma variegatum on a cattle population from Kenya (Mwangi et al., 1997). In Brazil, studies on tick parasitoids are scarce. For instance, Ixodiphagus species have been reported in R. sanguineus s.l. from the states of Rio de Janeiro (Costa-Lima, 1915), Maranhão (Lopes et al., 2012) and in Amblyomma spp. from the states of Mato Grosso do Sul and Rondônia (Lopes et al., 2012). Nevertheless, many aspects of Ixodiphagus life-history remain to be investigated, including study trials to assess the effectiveness of these parasitoids in the control of ticks, as well as their biology and geographical distribution. Therefore, the aim of this study was to report and molecularly characterize the occurrence of larvae of the genus Ixodiphagus parasitizing ticks in a tropical region of Brazil.
2. Material and methods 2.1. Study area and sampling The study was performed in the municipality of Garanhuns (Pernambuco state, northeastern Brazil, 8◦ 53 25 S and 36◦ 29 34 W). This region is 900 m above sea level, with an annual average tem-
http://dx.doi.org/10.1016/j.ttbdis.2017.03.004 1877-959X/© 2017 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Bezerra Santos, M.A., et al., Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.03.004
G Model
ARTICLE IN PRESS
TTBDIS-807; No. of Pages 3
M.A. Bezerra Santos et al. / Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
2
Fig. 1. Ixodiphagus sp. larva detected in the internal contents of Rhipicephalus sanguineus s.l. (Scale bar = 200 m).
perature of 24 ◦ C and annual relative humidity ranging from 80 to 100%. From October 2015 to March 2016, ticks were collected monthly from naturally infested dogs using forceps. Once collected, the specimens were placed into plastic vials and then morphologically identified (Walker et al., 2000).
2.4. Data analysis Data of positivity were assessed using chi-square tests with 95% confidence intervals. All analyses were performed using the software BioEstat 5.0 (Ayres et al., 2007). 3. Results
2.2. Tick dissection and wasp biology Specimens were dissected on a glass microscope slide with a drop of physiological solution (NaCl 0.9%) (Patton et al., 2012). Briefly, the hemolymph and intestinal contents of the ticks were exposed, the microscope slide was covered with a coverslip and examined at different magnifications (10 and 40X). Larvae were morphologically identified as belonging to the genus Ixodiphagus. In order to obtain adult wasps, a pool containing 10 engorged nymphs was stored in 50 ml polypropylene tubes at controlled conditions of temperature (28 ± 1 ◦ C) and relative humidity (80 ± 5%), and a natural day/night cycle for a period of 42 days (Tijsse-Klasen et al., 2011). The identification of adult wasps was performed following morphological features (Quaraishi, 1958).
2.3. Molecular procedures Larvae (n = 2) and adults (n = 2) were molecularly processed. DNA extraction was performed for both samples using a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, GmbH, Hidden, Germany) following the manufacturer’s instructions. Afterwards, a PCR targeting partial cytochrome c oxidase subunit 1 (cox1) gene sequence, using the primers (Ihookcox1F 5 -TTAGATGATTAGCTTCAATAAATGGAAT-3 and Ihookcox1R 5 CCAAAAATTGCAAAAACTGCTCCTAT-3 , 294 bp amplicon) designed from a partial I. hookeri cox1 gene sequence available in GenBank (JQ315225.1, Plantard et al., 2012) was performed (Ramos et al., 2015). Amplicons were sequenced in both directions using the same primers as used for PCR, employing Big Dye Terminator v.3.1 chemistry in a 3130 genetic analyzer (Applied Biosystems, CA, USA).
A total of 1814 R. sanguineus s.l ticks were collected from 83 naturally infested dogs. Of these, one specimen was an engorged larva, 263 engorged nymphs, 929 unfed adults, 194 partially engorged adults, and 427 fully engorged adults. During examination of the internal contents, larvae of parasitoids (n = 465) were detected by microscopic examination in 6.7% (n = 123) ticks. No statistical difference was observed between the positivity of partially engorged adults (23.7%; 46/194) and engorged nymphs (29.2%; 77/263) (x2 = 0.384; p = 0.5356). Morphometrically, larvae were 806.6 m (± 108.4) long and 175 m (± 16.5) wide (Fig. 1). A total of 21 adult wasps (5 males and 16 females) emerged from 2 engorged tick nymphs (mean = 10.55 adults/tick). All adults were morphologically identified as I. hookeri. At the PCR, all samples (larvae and adults) tested amplified a fragment of approximately 294 bp. The sequenced fragment (212 bp) showed 97% identity with I. hookeri sequences available from GenBank (accession numbers JQ315225). The sequence obtained in the present study was deposited in GenBank under accession number KY432404. 4. Discussion This study investigated the occurrence of larvae of the genus Ixodiphagus parasitizing ticks from a tropical region of Brazil. Parasitoid larvae were found in nymphs and partially engorged adults of R. sanguineus s.l. Nymphal stage ticks were more frequently found infected (29.2%) than partially engorged adults (23.7%), suggesting the tendency of the larvae of the genus Ixodiphagus to affect engorged nymphal ticks (Ramos et al., 2015). In fact, eggs of this wasp species develop more frequently in nymphs, moulting into a
Please cite this article in press as: Bezerra Santos, M.A., et al., Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.03.004
G Model TTBDIS-807; No. of Pages 3
ARTICLE IN PRESS M.A. Bezerra Santos et al. / Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
larval stage, which consumes the visceral contents of the engorged tick (Hu et al., 1998). However, parasitism in adult ticks cannot be discounted, since in a previous study a frequency of infection of 0.6% (3/481) was observed in adult I. ricinus (Ramos et al., 2015). This parasitoid was exclusively found in R. sanguineus s.l. because this was the only species collected in the study area, given that it was an urban area, where R. sanguineus s.l. is the most common tick species found (Dantas-Torres, 2009). Nevertheless, it is believed that other tick species may be affected by larvae of the genus Ixodiphagus in the region, as this wasp seems to have a worldwide distribution and wide host range (Hu et al., 1998). Larvae of the genus Ixodiphagus were only found in engorged nymphs and partially engorged adults, suggesting that parasitism is likely to occur more frequently in engorged ticks rather than in unfed specimens (Hu and Hyland, 1997). Nonetheless, studies using molecular tools suggest that unfed ticks, especially nymphs, may also be infested by larvae of the genus Ixodiphagus (Ramos et al., 2015). An experimental study, which assessed the parasitism in unfed and fully engorged nymphs and fully engorged larvae, demonstrated that unfed nymphs were more highly parasitized than the other life stages (Collatz et al., 2011). The DNA sequence obtained in the present study showed better alignment with an I. hookeri sequence found in I. ricinus in France (97% identity). Significant alignments were also observed with other species of wasps such as Ooencyrtus negerrimus (KP676606) and Cheiloneurus ceroplastis (KU499469), with an identity of less than 93%. The presence of this parasitoid in ticks from northeaster Brazil brings new possibilities to the alternative control of these arthropods. However, further studies on the biology of Ixodiphagus spp. are necessary in order to find ways to use it as an effective control method in the region and to study the possible environmental impact the use of those wasps can cause. In conclusion, results of this study demonstrate that larvae of the genus Ixodiphagus use engorged nymphs and partially engorged adult R. sanguineus s.l. ticks as a host. Further studies focusing on the identification of the species needs to be performed in order to clarify the effect of Ixodiphagus spp. on tick populations of the studied region. Acknowledgements Authors would like thank Victor Lima and Ana Gabriela (Federal Rural University of Pernambuco) for their support in the preparation of figure and data analysis, and Bronwyn Campbell (University of Bari) for her suggestions on the manuscript.
3
References Ayres, M., Ayres-Júnior, M., Ayres, D.L., Santos, A.A., 2007. BioEstat-Aplicac¸ões Estatísticas Nas áreas De Ciências Biomédicas. Ong Mamiraua, Belém, PA. Collatz, J., Selzer, P., Fuhrmann, A., Oehme, R.M., Mackenstedt, U., Kahl, O., Steidle, J.L.M., 2011. A hidden beneficial: biology of the tick-wasp Ixodiphagus hookeri in Germany. J. Appl. Entomol. 35, 351–358. Coronado, A., 2006. Ixodiphagus hookeri howard, 1907 (Hymenoptera: encyrtidae) in the brown dog tick rhipicephalus sanguineus latreille, 1806 (Acari: ixodidae) in Venezuela. Entomol. Trop. 21, 61–64. Costa-Lima, A., 1915. O chalcidideo, Hunterellus hookeri Howard, parasita do carrapato Rhipicephalus sanguineus Latreille, observado no Rio de Janeiro. Rev. Vet. Zootec. 5, 201. Dantas-Torres, F., 2009. Ticks on domestic animals in pernambuco, northeastern Brazil. Braz. J. Vet. Parasitol. 18, 22–28. Doube, B.M., Heath, A.C.G., 1975. Observations on the biology and seasonal abundance of an encyrtid wasp, a parasite of ticks in Queensland. J. Med. Entomol. 12, 443–447. Heath, A.C.G., Cane, R.P., 2010. A new species of Ixodiphagus (Hymenoptera: chalcidoidea: encyrtidae) parasitizing seabird ticks in New Zealand. N.Z. J. Zool. 37, 147–155. Hu, R., Hyland, K.E., 1997. Prevalence and seasonal activity of the wasp parasitoid, Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in its tick host, Ixodes scapularis (Acari: Ixodidae). Syst. Appl. Acarol. 2, 95–100. Hu, R., Hyland, K.E., Oliver, J.H., 1998. A review on the use of Ixodiphagus wasps (Hymenoptera: Encyrtidae) as natural enemies for the control of ticks (Acari: Ixodidae). Syst. Appl. Acarol. 3, 19–28. Lopes, A.J.O., Nascimento-Júnior, J.R.S., Silva, C.G., Prado, A.P., Labruna, M.B., Costa-Júnior, L.M., 2012. Parasitism by Ixodiphagus Wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus and Amblyomma Ticks (Acari: Ixodidae) in Three Regions of Brazil. J. Econ. Entomol. 5, 1979–1981. Mwangi, E.N., Hassan, S.M., Kaaya, G.P., Essuman, S., 1997. The impact of Ixodiphagus hookeri, a tick parasitoid, on Amblyomma variegatum (Acari: Ixodidae) in a field trial in Kenya. Exp. Appl. Acarol. 21, 117–126. Patton, T.G., Dietrich, G., Brandt, K., Dolan, M.C., Piesman, J., Gilmore, R.D., 2012. Saliva, salivary gland, and hemolymph collection from Ixodes scapularis Ticks. J. Vis. Exp. 60, 1–6. Plantard, O., Bouju-Albert, A., Malard, M.A., Hermouet, A., Capron, G., Verheyden, H., 2012. Detection of Wolbachia in the tick Ixodes ricinus is due to the presence ofthe hymenoptera endoparasitoid Ixodiphagus hookeri. PLoS ONE 7, e30692. Quaraishi, M.S., 1958. Morphology of two Chalcidoid parasites of ticks, Hunterellus hookeri Howard, 1908, and Ixodiphagus texanus Howard, 1907. Am. Mid. Nat. 59, 489–504. Ramos, R.A.N., Campbell, B.E., Whittle, A., Lia, R.P., Montarsi, F., Parisi, A., Dantas-Torres, F., Wall, R., Otranto, D., 2015. Occurrence of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Ixodes ricinus (Acari: Ixodidae) in Southern Italy. Ticks Tick-borne Dis. 6, 234–236. Tijsse-Klasen, E., Braks, M., Scholte, E.J., Sprong, H., 2011. Parasites of vectors − Ixodiphagus hookeri and its Wolbachia symbionts in ticks in the Netherlands. Parasit. Vectors 4, 228. Walker, J.B., Keirans, J.E., Horak, I.G., 2000. The genus rhipicephalus (Acari, ixodidae). In: A Guide to the Brown Ticks of the World. Cambridge University Press, Cambridge, United Kingdom, pp. 382–391. Wood, H.P., 1911. Notes on the life history of the tick parasite Hunterellus hookeri Howard. J. Econ. Entomol. 4, 425–431.
Please cite this article in press as: Bezerra Santos, M.A., et al., Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.03.004
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil Marcos Antônio Bezerra Santos a , Lucia Oliveira de Macedo a , Islanne Barbosa de Souza a , Carlos Alberto do Nascimento Ramos b , Leucio Câmara Alves c , Rafael Antonio Nascimento Ramos a,∗ , Gílcia Aparecida de Carvalho a a
Unidade Acadêmica de Garanhuns, Universidade Federal Rural de Pernambuco, Garanhuns, Brazil Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Campo Grande, Brazil c Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, Brazil b
a r t i c l e
i n f o
Article history: Received 10 January 2017 Received in revised form 12 March 2017 Accepted 15 March 2017 Available online xxx Keywords: Ticks Parasitoid Biological control Ixodiphagus hookeri
a b s t r a c t The biological control of ticks represents an alternative method to the chemical control, given its ecological-friendly approach. Amongst the alternatives, the use of parasitoids of the genus Ixodiphagus (Hymenoptera: Encyrtidae) has been largely investigated. The aim of this study was to document and molecularly characterize Ixodiphagus wasps in ticks from a tropical region of Brazil. From October 2015 to March 2016, Rhipicephalus sanguineus sensu lato ticks (n = 1814) were collected from naturally infested dogs and Ixodiphagus larvae were detected by microscopic examination. In addition, adult wasps were obtained in the laboratory. Larvae and adults were molecularly identified as Ixodiphagus hookeri. These findings suggest that this type of parasitism deserves to be studied in local tick populations, in order to elucidate the role of these wasps as a potential alternative to chemical tick control. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Ticks are amongst the most important vectors of a wide range of pathogens, including protozoa, rickettsiae, spirochetes, viruses and nematodes (Ramos et al., 2015; Hubálek and Rudolf, 2012). In addition, these ectoparasites may cause irritation, allergies, intoxication, and paralysis of their hosts. The control of these arthropods is of great importance for medical and veterinary science, given their global economic and public health impact. Over the last few years, the biological control of ticks using predators, parasitoids and arthropod pathogens has been investigated as an alternative to chemical control. Amongst these tools, parasitoids of the genus Ixodiphagus (Hymenoptera: Encyrtidae) have been documented in different tick species, such as Amblyomma spp. (Mwangi et al., 1997; Lopes et al., 2012), Ixodes spp. (Heath and Cane, 2010; Ramos et al., 2015), Dermacentor spp. (Wood, 1911), Haemaphysalis spp. (Doube and Heath, 1975) and Rhipicephalus sanguineus sensu lato (s.l.) (Coronado, 2006; Lopes et al., 2012). The efficacy of larvae of the genus Ixodiphagus in tick control is still debated among researchers since the biology of
∗ Corresponding author. E-mail address: [email protected] (R.A.N. Ramos).
these wasps has not yet been completely elucidated (Collatz et al., 2011). The most important study conducted so far, demonstrated the ability of this parasitoid to reduce the number of Amblyomma variegatum on a cattle population from Kenya (Mwangi et al., 1997). In Brazil, studies on tick parasitoids are scarce. For instance, Ixodiphagus species have been reported in R. sanguineus s.l. from the states of Rio de Janeiro (Costa-Lima, 1915), Maranhão (Lopes et al., 2012) and in Amblyomma spp. from the states of Mato Grosso do Sul and Rondônia (Lopes et al., 2012). Nevertheless, many aspects of Ixodiphagus life-history remain to be investigated, including study trials to assess the effectiveness of these parasitoids in the control of ticks, as well as their biology and geographical distribution. Therefore, the aim of this study was to report and molecularly characterize the occurrence of larvae of the genus Ixodiphagus parasitizing ticks in a tropical region of Brazil.
2. Material and methods 2.1. Study area and sampling The study was performed in the municipality of Garanhuns (Pernambuco state, northeastern Brazil, 8◦ 53 25 S and 36◦ 29 34 W). This region is 900 m above sea level, with an annual average tem-
http://dx.doi.org/10.1016/j.ttbdis.2017.03.004 1877-959X/© 2017 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Bezerra Santos, M.A., et al., Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.03.004
G Model
ARTICLE IN PRESS
TTBDIS-807; No. of Pages 3
M.A. Bezerra Santos et al. / Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
2
Fig. 1. Ixodiphagus sp. larva detected in the internal contents of Rhipicephalus sanguineus s.l. (Scale bar = 200 m).
perature of 24 ◦ C and annual relative humidity ranging from 80 to 100%. From October 2015 to March 2016, ticks were collected monthly from naturally infested dogs using forceps. Once collected, the specimens were placed into plastic vials and then morphologically identified (Walker et al., 2000).
2.4. Data analysis Data of positivity were assessed using chi-square tests with 95% confidence intervals. All analyses were performed using the software BioEstat 5.0 (Ayres et al., 2007). 3. Results
2.2. Tick dissection and wasp biology Specimens were dissected on a glass microscope slide with a drop of physiological solution (NaCl 0.9%) (Patton et al., 2012). Briefly, the hemolymph and intestinal contents of the ticks were exposed, the microscope slide was covered with a coverslip and examined at different magnifications (10 and 40X). Larvae were morphologically identified as belonging to the genus Ixodiphagus. In order to obtain adult wasps, a pool containing 10 engorged nymphs was stored in 50 ml polypropylene tubes at controlled conditions of temperature (28 ± 1 ◦ C) and relative humidity (80 ± 5%), and a natural day/night cycle for a period of 42 days (Tijsse-Klasen et al., 2011). The identification of adult wasps was performed following morphological features (Quaraishi, 1958).
2.3. Molecular procedures Larvae (n = 2) and adults (n = 2) were molecularly processed. DNA extraction was performed for both samples using a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, GmbH, Hidden, Germany) following the manufacturer’s instructions. Afterwards, a PCR targeting partial cytochrome c oxidase subunit 1 (cox1) gene sequence, using the primers (Ihookcox1F 5 -TTAGATGATTAGCTTCAATAAATGGAAT-3 and Ihookcox1R 5 CCAAAAATTGCAAAAACTGCTCCTAT-3 , 294 bp amplicon) designed from a partial I. hookeri cox1 gene sequence available in GenBank (JQ315225.1, Plantard et al., 2012) was performed (Ramos et al., 2015). Amplicons were sequenced in both directions using the same primers as used for PCR, employing Big Dye Terminator v.3.1 chemistry in a 3130 genetic analyzer (Applied Biosystems, CA, USA).
A total of 1814 R. sanguineus s.l ticks were collected from 83 naturally infested dogs. Of these, one specimen was an engorged larva, 263 engorged nymphs, 929 unfed adults, 194 partially engorged adults, and 427 fully engorged adults. During examination of the internal contents, larvae of parasitoids (n = 465) were detected by microscopic examination in 6.7% (n = 123) ticks. No statistical difference was observed between the positivity of partially engorged adults (23.7%; 46/194) and engorged nymphs (29.2%; 77/263) (x2 = 0.384; p = 0.5356). Morphometrically, larvae were 806.6 m (± 108.4) long and 175 m (± 16.5) wide (Fig. 1). A total of 21 adult wasps (5 males and 16 females) emerged from 2 engorged tick nymphs (mean = 10.55 adults/tick). All adults were morphologically identified as I. hookeri. At the PCR, all samples (larvae and adults) tested amplified a fragment of approximately 294 bp. The sequenced fragment (212 bp) showed 97% identity with I. hookeri sequences available from GenBank (accession numbers JQ315225). The sequence obtained in the present study was deposited in GenBank under accession number KY432404. 4. Discussion This study investigated the occurrence of larvae of the genus Ixodiphagus parasitizing ticks from a tropical region of Brazil. Parasitoid larvae were found in nymphs and partially engorged adults of R. sanguineus s.l. Nymphal stage ticks were more frequently found infected (29.2%) than partially engorged adults (23.7%), suggesting the tendency of the larvae of the genus Ixodiphagus to affect engorged nymphal ticks (Ramos et al., 2015). In fact, eggs of this wasp species develop more frequently in nymphs, moulting into a
Please cite this article in press as: Bezerra Santos, M.A., et al., Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.03.004
G Model TTBDIS-807; No. of Pages 3
ARTICLE IN PRESS M.A. Bezerra Santos et al. / Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
larval stage, which consumes the visceral contents of the engorged tick (Hu et al., 1998). However, parasitism in adult ticks cannot be discounted, since in a previous study a frequency of infection of 0.6% (3/481) was observed in adult I. ricinus (Ramos et al., 2015). This parasitoid was exclusively found in R. sanguineus s.l. because this was the only species collected in the study area, given that it was an urban area, where R. sanguineus s.l. is the most common tick species found (Dantas-Torres, 2009). Nevertheless, it is believed that other tick species may be affected by larvae of the genus Ixodiphagus in the region, as this wasp seems to have a worldwide distribution and wide host range (Hu et al., 1998). Larvae of the genus Ixodiphagus were only found in engorged nymphs and partially engorged adults, suggesting that parasitism is likely to occur more frequently in engorged ticks rather than in unfed specimens (Hu and Hyland, 1997). Nonetheless, studies using molecular tools suggest that unfed ticks, especially nymphs, may also be infested by larvae of the genus Ixodiphagus (Ramos et al., 2015). An experimental study, which assessed the parasitism in unfed and fully engorged nymphs and fully engorged larvae, demonstrated that unfed nymphs were more highly parasitized than the other life stages (Collatz et al., 2011). The DNA sequence obtained in the present study showed better alignment with an I. hookeri sequence found in I. ricinus in France (97% identity). Significant alignments were also observed with other species of wasps such as Ooencyrtus negerrimus (KP676606) and Cheiloneurus ceroplastis (KU499469), with an identity of less than 93%. The presence of this parasitoid in ticks from northeaster Brazil brings new possibilities to the alternative control of these arthropods. However, further studies on the biology of Ixodiphagus spp. are necessary in order to find ways to use it as an effective control method in the region and to study the possible environmental impact the use of those wasps can cause. In conclusion, results of this study demonstrate that larvae of the genus Ixodiphagus use engorged nymphs and partially engorged adult R. sanguineus s.l. ticks as a host. Further studies focusing on the identification of the species needs to be performed in order to clarify the effect of Ixodiphagus spp. on tick populations of the studied region. Acknowledgements Authors would like thank Victor Lima and Ana Gabriela (Federal Rural University of Pernambuco) for their support in the preparation of figure and data analysis, and Bronwyn Campbell (University of Bari) for her suggestions on the manuscript.
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Please cite this article in press as: Bezerra Santos, M.A., et al., Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.03.004