doi:10.12741/ebrasilis.v9i2.595 e-ISSN 1983-0572 Publicação do Projeto Entomologistas do Brasil www.ebras.bio.br Distribuído através da Creative Commons Licence v4.0 (BY-NC-ND) Copyright © EntomoBrasilis Copyright © do(s) Autor(es)

Predators of the Alfalfa Acyrthosiphon pisum (Harris), Aphis craccivora Koch, and trifolii (Monell) (: Aphidoidea) as Determined by the Serological Technique

Suzan Beatriz Zambon da Cunha¹, Carlos Roberto Sousa e Silva¹, Flávio Henrique Góis Diniz² & Evôneo Berti-Filho³

1. Universidade Federal de São Carlos, e-mail: [email protected] (Autor para correspondência), [email protected]. 2. Centro Universitário Central Paulista , e-mail: [email protected]. 3. Universidade de São Paulo, e-mail: [email protected]. ______

EntomoBrasilis 9 (2): 120-123 (2016) Abstract. The serology is an immunological technique based on antigen/antibody reactions in where its main advantages are high sensitivity and specificity that allows the biological recognition at the molecular level. This study evaluates the use of serology technique to determine the predators of the alfalfa aphids Acyrthosiphon pisum (Harris), Aphis craccivora Koch, and (Monell) (Hemiptera: Aphidoidea). The samplings to obtain the antibodies and their possible predators to be used as antigens were carried out in the alfalfa fields of the Embrapa Pecuária Sudeste Unit, São Carlos, SP. In the period from August 2011 to July 2012. A total of 2,161 predators, including and spiders, were tested. The antibodies obtained for the aphid A. craccivora, A. pisum, and T. trifolii showed partial identity nevertheless still allowed to recognize the predators of alfalfa aphids. Among the insects, syrphids and chrysopids presented the highest percentage of positive results in the serological tests. The species A. craccivora was the most preyed aphid.

Keywords: Antibodies; biological control; predation; serology.

Determinação de Predadores dos Pulgões da Alfafa Acyrthosiphon pisum (Harris), Aphis craccivora Koch e Therioaphis trifolii (Monell) (Hemiptera: Aphidoidea) por meio da Técnica Serológica

Resumo. A serologia é uma técnica imunológica baseada em reações antígeno/anticorpo, em que suas principais vantagens são a alta sensibilidade e especificidade que permitem o reconhecimento biológico em nível molecular. Este trabalho avalia o uso da técnica serológica para determinar os predadores dos pulgões da alfafa, Acyrthosiphon pisum (Harris), Aphis craccivora Koch e Therioaphis trifolii (Monell) (Hemiptera: Aphidoidea). As coletas dos pulgões para a obtenção dos anticorpos e de seus possíveis predadores para serem utilizados como antígenos foram realizadas nos campos de alfafa da Unidade da Embrapa Pecuária Sudeste, São Carlos, SP. no período de agosto de 2011 a julho de 2012. Foram testados 2.161 artrópodes predadores, incluindo insetos e aranhas. Os anticorpos obtidos para os pulgões A. craccivora, A. pisum, e T. trifolii mostraram identidade parcial mas, ainda assim, permitiu reconhecer os predadores dos pulgões da alfafa. Dentre os insetos, sirfídeos e crisopídeos foram os que apresentaram as maiores porcentagens de resultados positivos nos testes serológicos. A. craccivora foi o afídeo mais consumido pelos predadores.

Palavras-chave: Anticorpos; controle biológico; predação; serologia.

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lfalfa, Medicago sativa L., is one of the most cultivated (Neuroptera) and Syrphidae (Diptera) larvae are recognized as forage plants worldwide, being the United States, important aphid predators in alfalfa (Bu e n o & Si l v a 2008). Russia, Canada, and Argentina the main producers. The area cultivated with alfalfa in Brazil is around 30,000 ha, which Direct observation is theoretically the simplest method to is concentrated in the southern region and is responsible for determine the predatory actions, however, hardly applicable to 90% of total Brazilian production (Pe r e i r a 2008). The aphids small , such as , or those that are difficult to Acyrthosiphon pisum Harris (: Macrosiphini), Aphis approach (Da j o z 1973). craccivora Koch (Aphididae: Aphidini), and Therioaphis trifolii Immunoassay techniques like Serology and Polymerase Chain (Monell) (Drepanosiphidae: Phyllaphidini) are current pests of Reaction (PCR) show advantage over direct observation by alfalfa. They damage the alfalfa plants by shortening the plant minimizing researcher interference in the environment and by internodes, wrinkling the leaves, thus hindering the plant growth not ruling out the occurrence of nocturnal predators. Serology (Af o n s o 2008). is cheaper than PCR because it does not require sophisticated Outbreaks of alfalfa aphids are controlled by using chemical equipment and provides stable results and unlimited content of insecticides, which are dangerous not only to the environment, antibodies, allowing investigation of a large number of natural but also to alfalfa-feeding animals. Biological control is a good enemies (Ha r d w o o d & Ob r y c k i 2005). Entomologia Geral/ General Entomology alternative strategy to the use of chemicals; therefore it is very The serological technique is based on antigen / antibody reactions important to have knowledge on the occurrence of predators’ where the antibodies are obtained using the investigated aphids species of the alfalfa aphids. Among these predators adults and and the antigens obtained from the various aphid consumers. immature Geocoridae, Nabidae, Anthocoridae (Hemiptera: Heteroptera), Coccinellidae (Coleoptera) Chrysopidae

www.periodico.ebras.bio.br 121 9 nclto with inoculation 19 We with intervals. inoculations 10-day at 10 rabbit performed a in inoculated was macerate emulsified The (v/v). ratio a 1:1 at with adjuvant incomplete Freund’s emulsified supernatant the of mL 0.5 and discarded, was saline 0.85% a in 6,000 for fivemin.at and centrifuged solution macerated were samples the Thereafter, 24 h. for kept and separated were species aphid each of Samples serological reactions. AsTt, respectively, and the predators were used as antigens in the and aphids The 2012. July to 2011 August from Brazil, Paulo, São of state Carlos, São in Sudeste-CPPSE, Pecuária Embrapa of fields experimental in Crioula) (cv. plants group of predators used in this research were collected from alfalfa and AsTt(T.trifolii 1. Table were antiserum each from andits obtained titers first the The each antiserum after inoculation. days seven observed between were antigen results homologous positive first The aphids The predators inalfalfausingtheserologytechnique. craccivora A. aphids the from antisera produce to aimed study This according toageometricalprogressionratio2. performed were dilutions The AgTt. and AgAc, AgAp, antigens homologous respective their with reactions in antisera AsTt and by using fractions with 0.85 % NaCl solution of each AsAp, AsAc, performed were titers The slide). mm 25 x 75 per solution agar gel (O gel agar 1% in diffusion double by resulting performed were reactions These the and discarded antisera were were stored at -2 °C for later use in serological reactions. coagula the marginal temperature; the on room at h 2 for incisions remained sample Each ears. rabbit’s the of veins longitudinal small from were average) obtained on mL (10 samples blood The titers. their respective of evaluation and acquisition antisera for each in bloodsheds weekly performed we period, inoculation the During inoculations with Araneae Total Nabidae Dermaptera Geocoridae Pentatomidae Carabidae Coccinellidae (adults) Reduviidae Syrphidae Chrysopidae Coccinellidae (larvae) Predators oftheAlfalfaAphids Cycloneda sanguinea Harmonia axyridis Hippodamia convergens Eriopis connexa T. trifolii trifolii T. y n o l r e t h c u

A. pisum (A. AsAp craccivora), (A. AsAc antibodies the and alfalfa in collected predators among reactions serological from obtained Results A. pisum, A. craccivora, A. pisum, A. MATERIALS ANDMETHODS were used to obtain the antisera AsAp, AsAc, and AsAc, AsAp, antisera the obtain to used were and Predator

). 1958) with PBS buffer, pH 7.0, on slides (3 mL (3 slides on 7.0, pH buffer, PBS with 1958) (Germar) T. trifolii . trifolii T. (Pallas) . croccivora A. (L.) Guérin-Méneville RESULTS (AgTt)macerate. Acyrthosiphon pisum and use them to determine their their determine to them use and . pisum A.

and AA) aeae ad 11 and macerate, (AgAc) A. pisum, A. craccivora, A. pisum, A. T. trifolii T. AA) macerate, (AgAp) Number oftested . The precipitate g. The predators (Harris)… , as well as the as well as , 2161 1402 409 748 272 213 156 20 24 32 76 55 55 13 17 71 A. pisum, A. 4 species duringthecollectingperiod(Table2). consumed of number positive reactions with higher AsAc, that is, a with predators observed that probably We results. positive false to lead may antisera the among identity partial the Moreover, species. aphid three or two one, on preyed have may predator same the since results, positive of number total the to equal always not is We observed that the sum of positive results with each antiserum antiserum forT.trifolii cracivora, 296 with the antiserum for for antiserum the with results positive 392 showed predators The serological reactions between each antisera and the different (7.69%), Lycosidae Pholcidae (9.0%), Salticidae (7.14%),Thomisidae(6.75%),andCtenidae(5.28%). family Araneidae (14.28%), the Nephilidae by (Araneae), followed was and results, positive of 16.6% spiders with highlighted the Concerning lastly and %), (5.2 Dermaptera (7%). pentatomids nabids (27.2%), the geocorids by reduviids followed (33.9%), the results, positive Hemiptera, of the 41.1% represented Regarding results. positive of positive the convergens of 47.8% 22%; for responsible for were adults the while results, responsible were Coccinellidae of Larvae crop. alfalfa the in predators aphid as importance their chrysopids accounted for 65% of positive results, making evident The (67.2%). results positive of percentage highest the showed syrphids technique, The antiserum. aphid serological one least the at with by reacted 23.5% tested predators 508 the From Pholcidae, Nephilidae, Trochanteriidae wereidentified. Linyphiidae, and Thomisidae, Theridiidae, Sparassidae, Sicariidae, Salticidae Lycosidae, Araneidae, families Ctenidae, the of specimens spiders, the for whereas and Pentatomidae, recorded; were (Chrysopidae) Neuroptera and Reduviidae), and Nabidae, (Dolichopodidae (Geocoridae, Hemiptera Diptera Syrphidae), and Dermaptera, (Carabidae Coleoptera of Coccinellidae), specimens spiders: We insects, and the identity. insects among partial predaceous of a specimens showing 2,161 collected aphids, alfalfa with different reacted also the antiserum each with reactions homologous /2 o AA, /2 o AA, n 18 o AT, fe te 3 the after AsTt, for 1/8 and AsAc, for 1/32 AsAp, for 1/32 positive results th , and 5 and , Number of 508 309 136 127 38 34 32 53 37 13 15 8 4 3 7 1 th A. craccivora, inoculation in each rabbit, respectively. In addition, In respectively. rabbit, each in inoculation Guérin-Méneville stood out in this group with 30.5% (Table1). AsAc 392 231 even this aphid being the less abundant 84 36 65 74 27 41 21 11 11 8 3 3 7 1 Results witheachantiserum A. pisum AsAp 296 178 38 29 10 81 61 31 13 15 e-ISSN 1983-0572 0 0 8 2 3 5 ,

and 224 with the Cunha etal. Hippodamia AsTt 224 108 28 46 24 29 23 27 21 0 9 4 4 3 5 1 A. rd ), , Maio - Agosto 2016 - www.periodico.ebras.bio.br EntomoBrasilis 9 (2)

Table 2. Number of aphids sampled for each species in alfalfa from presenting the highest frequency of positive serological results. August 2011 to July 2012. Highest frequencies of positive serological reactions occurred Period A. pisum A. craccivora T. trifolii with the antiserum obtained for A. craccivora (AsAc). Spring 491 430 1658 ACKNOWLEDGEMENTS Summer 3734 1377 2056 We are grateful to Conselho Nacional de Desenvolvimento Autumn 1228 227 566 Científico e Tecnológico (CNPq) for financial support. Winter 314 535 494 REFERENCES

DISCUSSION Afonso, A.P.S., 2008 Insetos pragas da alfafa, p. 18-33. In: Mittelmann, A., F.J.S. Ledo & J.F. Gomes (Eds.). Tecnologias Among the tested predators, syrphids stood out for presenting para produção de alfafa no Rio Grande do Sul. EMBRAPA the highest frequency of positive results. Syrphids are considered – Centro de Pesquisa Agropecuária de Clima Temperado, to be essential predators of aphids in Portugal (Il h a r c o 1992), as Pelotas RS/ Juiz de Fora MG. 70 p. well as potential predators of many aphid species with economic Bastos, C.S. & J.B. Torres, 2005. Controle biológico e manejo de importance foragricultural crops in Brazil (Ba s t o s & To r r e s 2005). pragas do algodoeiro. Campina Grande: Embrapa. 63 p. Chrysopids also highlighted for the high percentage of positive Bueno, V. H.P. & A.C. Silva, 2008. Pragas na cultura de alfafa, results, being surpassed only by the syrphids. According to Ba s t o s p. 289-316. In: Ferreira, R.P., J.B. Rassini, A.A. Rodrigues, & To r r e s (2005), although being able to prey on a diverse array of A.F. Freitas, A.C. Camargo & F.C. Mendonça (Eds.). Cultivo species, chrysopids prey preferably on aphids; during their e utilização da alfafa nos trópicos, São Carlos, Embrapa larval stage chrysopids can consume from 100 to 600 aphids. Pecuária Sudeste. 326 p. The coccinellids stand out as aphid predators (Sa r a n et al. 2007) Dajoz, R., 1973. Ecologia Geral. Petrópolis, Editora Vozes, 474 p. and, even without any species of this exclusively afidophagous Giorgi, J.A., N.J. Vandenberg, J.V. Mchung, J.A. Forrester, A. family, some have strong preferences for these insects, such as Slipinski, K.B. Miller, L.R. Shapiro & M.F. Whiting, 2009. Cycloneda, Harmonia and Hippodamia (Gi o r g i et al. 2009), as The evolution of food preferences in Coccinellidae. Biological observed in the present study. Control, 51: 215-231. DOI: http://dx.doi.org/10.1016/j. Ka t o et al. (1999) reported that H. convergens is responsible biocontrol.2009.05.019. for maintaining alfalfa aphid populations below the threshold Greenstone, M.H. & J.H. Hunt, 1993. Determination of prey levels in California. Sa r a n et al. (2007) stated that larvae of antigen half-life in Polistes metricus using a monoclonal some coccinellid species have a daily prey intake higher than antibody-based immunodot assay. Entomologia that showed by the adults. Daily consumption of Cycloneda Experimentalis et Applicata, 68: 1-7. DOI: http://dx.doi. sanguinea (L.) larva, for instance, may reach 200 aphids, while org/10.1111/j.1570-7458.1993.tb01682.x. the adult consumes only 20 aphids per day on average. Hardwood, J.D. & J. Obrycki, 2005. Quantifying aphid predation rates of generalist predators in the field. European Journal of Spiders are generalist predators. Besides using the food that is Entomology, 102: 335-350. DOI: http://dx.doi.org/10.14411/ trapped in their webs they are also avid hunters (Sa r a n et al. eje.2005.051. 2007). In the present study spiders were observed both in the soil Ilharco, F.A., 1992. Equilíbrio biológico de afídeos. Lisboa, and in the higher parts of the alfalfa plant. Gr e e n s t o n e & Hu n t Fundação Calouste Gulbenkian. 300 p. (1993) stated that serology is an important method to studying Kato, C.M., V.H.P. Bueno, J.C. Morais & A.M. Auad, 1999. Criação preying by spiders; because only the prey’s body fluids are de Hippodamia convergens Guérin-meneville (Coleoptera: consumed by spiders, it is difficult to identify the prey using other Coccinellidae) em ovos de Anagasta kuehniella (Zeller) techniques. Su n d e r l a n d et al. (1987) mentioned Lycosidae and (Lepidoptera: Pyralidae). Anais da Sociedade Entomológica Tetragnatidae as important families of aphid predaceous spiders do Brasil, 28: 455-459. DOI: http://dx.doi.org/10.1590/ in cereal crops. Although Il h a r c o (1992) reported some evidence S0301-80591999000300010. that spiders might prey on aphids, little interest has been shown Ouchterlony, O., 1958. Diffusion in gel methods for immunological to this group as biological control agents of aphids. analysis, p. 1-78/11 In: S. Karger (Ed.). Progress in allergy. New York, Plenum Press. 312 p. According to Su n d e r l a n d et al. (1987), the high variation in aphid Pereira, A.V., 2008. Cultivares de alfafa, p. 289-316. In: Ferreira, detection by using the serological technique may be attributed R.P., J.P. Rassini, A.A. Rodrigues, A.F. Freitas, A.C. Camargo to the prey size, and even to the predator size. The aphids A. & F.C. Mendonça (Eds.). Cultivo e utilização da alfafa nos craccivora and T. trifolii have similar body sizes, varying from trópicos. São Carlos, Embrapa Pecuária Sudeste. 469 p. 1.4 to 2.2 mm lenght. Such slight variation in body size would not Saran, E.D., D. Thomazoni, A.P. Serra & P. Degrande, 2007. be the only factor responsible for prey choice by the predators. Manual dos insetos benéficos do algodoeiro. Campinas, FMC A. pisum body size ranges from 2.5 to 4.4 mm, and in spite of - Química do Brasil. 227 p. the higher body size, this aphid was not the least preyed species Sunderland, K.D., N.E. Crook, D.L. Stacey & B.J. Fuller, 1987. A among the tested aphids. These results indicate that besides the study of feeding by polyphagous predators on cereal aphids body size, other factors, such as prey palatability and toxicity, using Elisa and gut dissection. Journal of Applied Ecology, may influence predator searching and choice for most suitable 24: 907-933. DOI: http://dx.doi.org/10.2307/2403989. preys.

The serology, being a highly sensitive and specific technique Received in: 20.iii.2016 can be successfully used to qualify predators in studies of food Accepted in: 04.vi.2016 relationships, especially taking into account small organisms ********** such as arthropods. In biological control programs it allows to estimate the preference of a given predator for a certain prey.

In conclusion, the antisera obtained for the aphids A. craccivora, 122 A. pisum, and T. trifolii showed partial identity. It was possible to recognize the alfalfa aphid predators by using the serology technique. Syrphids and chrysopids were the predators

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Suggestion citation:

Cunha, S.B.Z. da, C.R. Sousa e Silva, F.H.G. Diniz & E. Berti-Filho, 2016. Predators of the Alfalfa Aphids Acyrthosiphon pisum (Harris), Aphis craccivora Koch, and Therioaphis trifolii (Monell) (Hemiptera: Aphidoidea) as Determined by the Serological Technique. EntomoBrasilis, 9 (2): 120-123. Available on: doi:10.12741/ebrasilis.v9i2.595 123

e-ISSN 1983-0572