UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS – RIO CLAROunesp PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (ZOOLOGIA) BIOLOGIA, MORFOLOGIA, E BIOQUÍMICA DE VENENO DA FORMIGA LAVA-PÉS Solenopsis saevissima Smith (INSECTA: HYMENOPTERA: FORMICIDAE) EDUARDO GONÇALVES PATERSON FOX Tese apresentada ao Instituto de Biociências do Câmpus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas (área de concentração: Zoologia). Abril - 2010 BIOLOGIA, MORFOLOGIA E BIOQUÍMICA DE VENENO DA FORMIGA LAVA-PÉS Solenopsis saevissima Smith (INSECTA, HYMENOPTERA, FORMICIDAE) EDUARDO GONÇALVES PATERSON FOX Orientador: Prof. Dr. Odair Correa Bueno Tese apresentada ao Instituto de Biociências do Campus de Rio Claro, Universidade Estadual Paulista Júlio de Mesquita Filho, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas (Área de Zoologia) Rio Claro Estado de São Paulo – Brasil Abril de 2010 Fox, Eduardo Gonçalves Paterson Biologia, morfologia e bioquímica de veneno da formiga lava-pés, Solenopsis saevissima Smith (Insecta: Hymenoptera: Formicidae) / Eduardo Gonçalves Paterson Fox. - Rio Claro : [s.n.], 2010 123 f. : il., figs., tabs. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Rio Claro Orientador: Odair Correa Bueno 1. Formiga. 2. Formigas - História natural. 3. Formiga de fogo. 4. Toxina. 5. Formigueiro. 6. Larva. 7. Alcalóide. I. Título. 595.796 F791b Ficha Catalográfica elaborada pela STATI - Biblioteca da UNESP Campus de Rio Claro/SP i Ao meu avô, que sempre serviu de modelo para todos que o cercaram durante a vida. ii AGRADECIMENTOS Aos meus orientadores por terem me ajudado neste longo e difícil processo de investigação. Aos colaboradores desta pesquisa, provenientes de diversas instituições (em especial CEIS/UNESP, NAP/NEPA/ESALQ, CENA/USP, IQ/UNICAMP, MZUSP, IBCCF/UFRJ) que participaram de cada momento das descobertas em suas respectivas áreas, contribuindo para a formação do panorama geral como aqui apresentado. Aos colegas de instituição pelos momentos de trabalho e lazer proporcionados, bem como ajuda nas coisas mais simples mas que são tão importantes. Aos amigos e companheiros que ganhei me deslocando de longe para outra cultura, pela troca de idéias e momentos prazerosos. Aos amigos mais chegados, por definição tão poucos e tão valiosos, pelos momentos inesquecíveis juntos e por nunca terem me abandonado nas horas mais difíceis. Finalmente, agradeço a todos aqueles que de alguma forma estiveram presentes em minha vida nestes últimos anos. Todos de alguma forma contribuem para os resultados aqui apresentados e para tudo que for acontecer de aqui para adiante, no verdadeiro início da minha vida profissional. A Deus, que sempre me garantiu a vitória sobre os maiores desafios. iii “This species [Solenopsis saevissima] is exclusively found in sandy soils, in open semi-cultivated or neglected places […] they increase only in the neighbourhood of deserted houses or unweeded plantations; consequently they are a scourge only to the lazy and worthless people that inhabit the shores of this magnificent river.” Henry Bates, O Naturalista no Rio Amazonas (1855). Escrito cerca de 100 anos antes das formigas lava-pés se tornarem uma das piores pragas do mundo. iv RESUMO A formiga lava-pés Solenopsis saevissima Smith está entre os insetos que mais causam acidentes no Brasil, e é uma espécie pouco estudada. A presente série de investigações tenta suprir um pouco da necessidade de estudos com esta importante espécie no Brasil. Primeiramente são relatados detalhes da biologia de S. saevissima em comparação com outras espécies de formigas lava-pés: pela primeira vez é mostrada uma lista de artrópodes associados a estes formigueiros no Brasil, incluindo uma série de novos táxons, dos quais um é aqui descrito; as larvas desta espécie são descritas e comparadas com o que se sabe sobre as larvas de outras lava-pés, sendo visto que as semelhanças encontradas são extensas demais para permitir a utilização de caracteres larvais para filogenia e taxonomia em nível de espécie. Ainda na morfologia, são apresentados resultados de análise ultraestrutural do aparato de veneno por meio de microscopia ótica e eletrônica, onde é mostrado que as diferentes regiões do órgão apresentam especializações para a produção de cada um dos compostos do veneno. A composição do veneno desta espécie foi analisada pela primeira vez, onde verificou-se que acima de 90% do veneno de S. saevissima é composto de isômeros cis e tras de um mesmo alcalóide piperidinico oleoso, sendo o restante uma solução aquosa de toxinas protéicas, incluindo neurotoxinas, fosfolipases, e alérgenos. De uma forma geral, o veneno de S. saevissima tem uma diversidade menor de compostos que o de Solenopsis invicta, podendo figurar entre os motivos que explicam porque a espécie S. invicta é uma espécie invasora e S. saevissima não. São apresentados pela primeira vez evidências químicas da existência de espécies crítpticas dentro de S. saevissima. Tomados em conjunto, os resultados suprem um pouco da carência de estudos com as formigas lava-pés na América do Sul e demonstram a diversidade de assuntos ainda a serem investigados nestes insetos. Palavras-chave: formiga-de-fogo, taxonomia, sistemática, toxinologia, artrópode peçonhento, toxina. v ABSTRACT The fire ant Solenopsis saevissima Smith is one of the insects most frequently involved in accidents in Brazil, yet being a poorly studied species. The series of studies presented here aimed at filling some of this gap in knowledge about this common and important ant species. Some aspects of the field biology of S. saevissima are shown in comparison with other fire ants: a unique list of associated arthropods collected from field inspections in Southern Brazil is given, which includes several new taxa, one of which is herein described for the first time. The larvae of S. saevissima are described for the first time and compared with larvae from close species, culminating with the demonstration that larval characters within this group cannot be feasibly employed in species-level phylogenetic and taxonomic analyses. In terms of internal anatomy, a detailed ultrastructural description of the venom apparatus of S. saevissima is given, wherein special emphasis was given to the particular organisation of each region of the apparatus, suggesting there are specialised areas for the production of each venom compound. The venom of this species was subject of biochemical analyses for the first time, generally illustrating that the venom of S. saevissima is >90% made of a simple mixture of cis- and tras- undecil-pyperidinic alkaloids, being the remainder an aqueous solution of toxic proteins, comprising neurotoxins, and traces of phospholipases and allergens. The venom of S. saevissima proved being less diverse in toxins than the venom of Solenopsis invicta, possibly explaining why S. invicta is a successful invasive species while S. saevissima apparently is not. Moreover, herein is included the first record of intraspecific variation in the nature of venom alkaloids, providing biochemical evidence for the existence of cryptic species in S. saevissima. Taken together, the obtained results contribute to the body of knowledge about fire ant populations in South America, and are proof of the existence of paramount facets yet to be investigated in deeper details. Keywords: fire ant, taxonomy, systematics, venom toxins, venomous arthropod, morphology. vi Organização da tese Esta tese teve como objetivo geral apresentar resultados sobre vários aspectos da biologia e bioquímica de veneno das formigas lava-pés, em especial S. saevissima. Estes resultados foram aqui agrupados em capítulos individualizados de acordo com o assunto de que tratam. Cada capítulo já foi escrito e organizado em formato de publicação, logo todos estão no idioma internacional inglês e incluem resumo, introdução, discussão e conclusões próprias. Ao fim da tese, panorama geral sobre as partes é traçado para que se possa avaliar o que foi obtido no conjunto, e uma série de perspectivas futuras são delineadas. O capítulo 1 apresenta uma lista de artrópodes inquilinos encontrados no interior dos formigueiros de lava-pés durante as coletas no campo, bem como faz comentários sobre a distribuição das espécies nas áreas investigadas. É enfatizada a carência de estudos de biologia geral com as formigas lava-pés no Brasil, inclusive constando na lista um grande número de espécies de artrópodes desconhecidas ou raramente encontradas na literatura. O capítulo 2 apresenta uma descrição morfológica detalhada de uma destas novas espécies, pertecente a um novo gênero de tisanuros (traças) do Brasil. O capítulo 3 apresenta a morfologia de todos os estádios imaturos de S. saevissima com imagens detalhadas de microscopia eletrônica de varredura, e discute as características observadas em comparação com outras espécies para determinar a relevância para a taxonomia do grupo. O capítulo 4 aborda a estrutura do aparato de veneno e de cada uma de suas partes, em comparação com o que foi feito com outras espécies de formigas lava- pés. O capítulo 5 relata os resultados sobre os alcalóides de veneno e hidrocarbonetos cuticulares obtidos para S. saevissima, bem como fornece fortes evidências da existência de espécies crípticas, ilustrando como maiores estudos podem influenciar a sistemática atual do grupo. O capítulo 6 apresenta uma análise proteômica do veneno das formigas lava- pés, obtida com base em um novo método de extração de veneno em grande quantidade desenvolvido durante as investigações da tese. O resultados aqui apresentados são os primeiros resultados de análise proteômica do veneno de uma formiga. vii As conclusões gerais são apresentadas ao final da tese em cima das conclusões de cada capítulo, unindo as informações para formar uma visão geral e enunciar as perspectivas futuras das investigações que estão sendo feitas em cada área. SUMÁRIO Página Introdução Objetivos Capítulo 1. Capítulo 2. Capítulo 3. Capítulo 4. Capítulo 5. Capítulo 6. Conclusões Gerais Perpectivas Futuras ............................................................................................ ............................................................................................ Uma lista preliminar dos inquilinos encontrados dentro de formigueiros de lava-pés no Sudeste do Brasil.................. Sobre um novo Nicoletiidae (Zygentoma: Insecta) do Brasil vivendo com formigas lava-pés (Hymenoptera: Formicidae)......................................................................... Sobre as larvas da formiga lava-pés Solenopsis saevissima. ........................................................................ Morfologia geral e ultraestrutural do aparato de veneno da formiga lava-pés Solenopsis saevissima...................... Caracterização dos alcalóides de veneno e hidrocarbonetos cuticulares da formiga lava-pés Solenopsis saevissima....................................................... Sobre as proteínas de veneno das formigas lava-pés: Análise proteômica do veneno de Solenopsis invicta e Solenopsis saevissima....................................................... ............................................................................................ ............................................................................................ 8 13 21 29 44 64 83 102 120 122 8 Introdução As formigas lava-pés O gênero Solenopsis inclui cerca de 277 espécies (BOLTON, 2006) de ocorrência mundial, sendo que umas vinte destas são espécies maiores e mais agressivas conhecidas como “formigas lava-pés” ou “formigas de fogo”, por causa da dor causada por suas ferroadas. Estas formigas são únicas entre os artrópodes por possuirem uma mistura de alcalóides em seus venenos aliados a uma pequena quantidade de proteínas alergênicas, sendo esta combinação responsável pelos incômodos gerados pelas ferroadas. As formigas lava-pés apresentam ampla ocorrência no território brasileiro, inclusive dentro das zonas urbanas, onde ocorrem principalmente nas beiras de estradas e gramados. As formigas lava-pés são onívoras e oportunistas, que podem predar tanto vertebrados e invertebrados quanto plantas (VINSON, 1994), além de terem o hábito de complementar sua dieta com secreções provenientes de outros invertebrados como, por exemplo, os insetos sugadores de seiva conhecidos como afídios (GREEN, 1952). Os ninhos são construídos diretamente no chão, em áreas abertas e ensolaradas, constituídos basicamente de um monte de terra no interior do qual fica a colônia, da qual irradiam vários túneis de forrageio (PORTER; TSCHINKEL, 1987, ver Figura 1). Devido à sua agressividade, proximidade dos ninhos das habitações humanas, e ao hábito de se associar a insetos sugadores de seiva, uma série de problemas são gerados pela presença das formigas lava-pés, que vão desde acidentes com animais e populações até estragos gerados na agricultura (LOFGREN et al., 1975). Algumas espécies de lava-pés foram acidentalmente transportadas a partir do Brasil para outras partes do mundo através de navios carregando madeira (TABER, 2000). Dentre estas, a espécie Solenopsis invicta Buren é a que causa mais estragos em todos os países em que se estabeleceu, gerando graves problemas de saúde e agrícolas com prejuízos elevados, sendo atualmente um dos insetos invasores mais importantes do mundo (HENSHAW et al., 2005). As espécies de formigas lava-pés mais comuns no Brasil são S. invicta e Solenopsis saevissima Smith, que podem ser encontradas em diversas regiões do 9 país (ROSSI; FOWLER, 2004). Apesar de causar muitos acidentes na região Amazônica, sendo considerada uma séria praga em algumas localidades (LUNZ et al., 2009), a espécie S. saevissima nunca foi registrada como invasora em outros países. Como uma consequência de sua importância social mais restrita, esta espécie não é bem conhecida e estudada como é a espécie invasora S. invicta, havendo uma grande carência de conhecimento em vários aspectos de sua biologia. A problemática na distinção entre espécies de formigas lava-pés As espécies de formigas lava-pés são difíceis de se determinar por morfologia devido aos caracteres serem variáveis e inconspícuos, além de haver um número ainda indeterminado de espécies intercruzantes (PITTS et al., 2005; VANDER MEER; LOFGREN, 1985; TRAGER, 1991). As diferentes espécies de formigas lava- pés são todas polimórficas e de morfologia bastante semelhante, sendo este grupo considerado um dos mais controversos quanto à sistemática e a filogenia (PITTS et al. 2005). Os caracteres morfológicos empregados na separação das espécies são de difícil observação e a morfologia destes caracteres apresenta um grau considerável de variação intraespecífica, e às vezes, dentro de uma mesma colônia (PITTS et al., 2005, vide alguns caracteres na Figura 2). A problemática é tornada mais difícil pela existência de espécies intercruzantes e até de espécies não descritas (PITTS et al., 2005). Sendo assim, a identificação da espécie de uma amostra de lava-pés depende da opinião de um especialista experiente com o grupo, sendo não raro impossível, dependendo das condições da amostra. Acredita-se que ferramentas moleculares tais como aloenzimas, marcadores de mt-RNA, determinação de hidrocarbonetos de cutícula e composição bioquímica de venenos, possam auxiliar grandemente na separação de espécies dentro deste grupo de difícil classificação (VANDER MEER; LOFGREN, 1998; STEINER et al., 2002; ROSS; SHOEMAKER, 2005). Uma destas ferramentas são os hidrocarbonetos cuticulares, satisfatoriamente já aplicados na separação entre S. invicta e Solenopsis richteri Forel 1923, duas espécies muito semelhantes, tendo sido a mesma ferramenta utilizada para demonstrar que há hibridização entre estas duas espécies (VANDER MEER; LOFGREN, 1985). Há autores que afirmam que também uma classe de compostos abundantes no veneno das formigas lava-pés, denominados de alcalóides, podem ser ferramentas úteis na sistemática deste grupo (GORMAN et al., 1998; VANDER 10 MEER; LOFGREN, 1985; DALL’AGLIO-HOLVORCEM, 2006; Figura 4), uma vez que as espécies mais estudadas apresentaram padrões de alcalóides de veneno distintos e específicos. Recentemente, um estudo demonstrou a utilidade dos hidrocarbonetos e destes na distinção entre populações de S. invicta e S. saevissima dentro do Estado de São Paulo, Brasil (DALL’AGLIO-HOLVORCEM et al., 2009). Como as proteínas de veneno são, em princípio, mais difíceis de se obter por estarem presentes em quantidades diminutas, e não foram estudadas em diferentes espécies, sua aplicabilidade na sistemática e taxonomia ainda permanece uma incógnita. No tocante ao caso específico da espécie-alvo do presente estudo, S. saevissima, um artigo recente apontou a existência de mais de um haplótipo dentro desta espécie baseado na estrutura molecular de populações de S. saevissima de diversas regiões da América do Sul, sugerindo a existência de espécies crípticas. Estas espécies são morfologicamente idênticas, porém podem ter características fisiólogicas distintas, como por exemplo, a composição de venenos. De posse desta informação, a presente investigação restringiu as análises e estudos às populações de S. saevissima de uma única região geográfica fixa, onde as amostras coletadas apresentassem os mesmos alcalóides de veneno. O veneno das formigas lava-pés Os constituintes do veneno dos insetos himenópteros são produzidos por duas glândulas exócrinas anexas ao ferrão: a glândula ácida (ou glândula de veneno) e a glândula básica (ou glândula de Dufour) (CRUZ-LANDIM; ABDALLA, 2002; BILLEN et al., 2000). O conjunto destas glândulas e mais o reservatório de veneno é denominado de aparato de veneno. Conforme mencionado anteriormente e discutido em maiores detalhes adiante, o veneno das formigas lava-pés é uma mistura de uma grande quantidade de alcalóides (>90%) com uma solução aquosa de proteínas alergênicas. A presença das lava-pés perto das habitações humanas freqüentemente causa acidentes. Estima-se que de aproximadamente 1.500 acidentes oficialmente registrados ao ano com formigas no Estado de São Paulo, acima de 30% sejam provenientes de ferroadas de formigas lava-pés (comunicação pessoal do Prof. Dr. MÁRIO SÉRGIO PALMA), em especial S. saevissima. Uma única colônia destes insetos costuma ter milhares de indivíduos armados com ferrões. As formigas 11 atacam agarrando-se firmemente à pele da vítima com as mandíbulas e ferroando repetidas vezes, em um padrão de movimento circular (HOFFMAN, 1995). Geralmente as ferroadas causam reações desagradáveis passageiras, como dor no momento da picada (reação atribuída aos alcalóides) seguida de queimação e forte coceira local (reação atribuida aos alérgenos). A maioria dos acidentes ocorre por contato direto com o formigueiro, onde uma grande quantidade de formigas pode estar envolvida no acidente. Dependendo do número de ferroadas e da sensibilidade da vítima aos compostos do veneno, a situação do paciente pode evoluir para quadros mais graves, como coceira pelo corpo inteiro, inchaço do membro atacado, necrose de tecido e até choque anafilático seguido de dificuldade de respiração, estado de coma ou morte (DESHAZO et al., 1984; STABLEIN; LOCKEY, 1987; RHOADES et al., 1989; PRAHLOW; BARNARD, 1998). A maioria das informações sobre os venenos das lava-pés é originária de estudos norte-americanos com a espécie invasora S. invicta, devido à sua importância local conforme comentado. Também existem alguns estudos sobre poucas outras espécies de formigas lava-pés (BLUM et al., 1961; CRUZ-LOPES et al. 2001; BLUM et al., 1974; JONES et al. 1996; JONES; BLUM, 1982). No Brasil as espécies S. invicta e S. saevissima são as mais abundantes, e há locais onde inclusive há predominância de S. saevissima (ROSSI e FOWLER, 2004; LUNZ et al., 2009). Sendo assim, uma grande parte dos acidentes com himenópteros no Brasil são causados pela espécie S. saevissima, porém não há nenhum estudo na literatura sobre o veneno desta espécie. Os alcalóides de veneno das formigas lava-pés foram alvo de uma série de estudos (MACCONNELL et al., 1970, 1971; JONES et al., 1982; BLUM et al., 1992; LECLERCQ et al., 1994; CHEN; FADAMIRO, 2009), onde foi verificado que se trata de uma mistura (na maioria das espécies) de 2-methyl-6-alkylpiperidinas. O número de carbonos presentes na cadeias laterais destes compostos e o estado de saturação é utilizado na representação por escrito destes compostos, que usualmente também recebem nomes informais (p.ex. trans-C11:1 se refere a uma piperidina em configuração espacial trans com onze carbonos na cadeia lateral com uma ligação dupla, sendo também conhecida como isosolenopsina). Algumas espécies possuem apenas formas cis e trans de uma única piperidina, mas a maioria apresenta uma mistura complexa de piperidinas que variam em comprimento de C11 a C15 (MACCONNELL et al., 1970). Alguns destes alcalóides demonstraram 12 atividade antifúngica e antimicrobiana, além de outras pronunciadas atividades biológicas sobre o sistema circulatório, nervoso e imune de vertebrados (vide HOWELL et al., 2005). Uma ferroada de uma formiga lava-pé injeta cerca de 20nl contendo 10-100ng de proteína (HOFFMAN et al., 1988). O veneno possui apenas 0,1-1% (p/v) de uma solução aquosa de proteínas (BAER et al., 1979), sendo que o restante (acima de 90%) do veneno se consiste de alcalóides piperidinas insolúveis em água (n-alquil e alcenil). Os alcalóides são responsáveis pela queimação e formação de pústulas na ferroada (JUNG et al., 1963), enquanto que as proteínas geram as reações alérgicas que podem variar de intensidade conforme a sensibilidade da vítima. As proteínas de veneno das formigas lava-pés figuram entre os alérgenos mais potentes do mundo (SCHMIDT et al., 1998), porém poucos estudos foram feitos com estas proteínas devido à sua ínfima quantidade no veneno e às dificuldades de se obter veneno de formigas em grandes quantidades. Ainda assim, foram isolados e caracterizados quatro alérgenos do veneno de S. invicta (HOFFMAN et al., 1988; HOFFMAN, 1993a), chamados de Sol i 1, Sol i 2, Sol i 3 e Sol i 4. O alérgeno Sol i 1 é a proteína de maior peso molecular (~34kDa) e está presente em menor quantidade no veneno (de 2-5%); possui atividade como fosfolipase A1B (HOFFMAN et al. 1988). O alérgeno Sol i 3 costuma ser bastante abundante (67%), sendo um dímero de 26kDa formado de dois monômeros de 13kDa. Os demais alérgenos Sol i 2 e Sol i 4 formam juntos cerca de 15-20% do veneno e ambos possuem cerca de 15kDa de peso molecular (HOFFMAN, 1993b). A literatura científica reporta o estudo das proteínas de veneno de apenas uma outra espécie de formiga lava-pes, Solenopsis richteri, que possui compostos bastante similares, porém com algumas diferenças na sequência dos aminoácidos e estando o equivalente ao alérgeno Sol i 4 ausente (HOFFMAN et al., 1990). 13 OBJETIVOS Objetivo Geral: Agregar conhecimento sobre a biologia, morfologia e bioquímica de veneno de S. saevissima, dado que esta foi pouco estudada até o momento. Objetivos específicos: • Levantar os dados biológicos e morfólogicos existentes na literatura sobre S. saevissima. • Obter dados de biologia de campo de S. saevissima • Descrever os estádios imaturos de S. saevissima por meio de análise por micoscopia ótica e de varredura. • Descrever o aparato de veneno de operárias de S. saevissima utilizando histologia e micrografias óticas e eletrônicas de transmissão e varredura. • Investigar os alcalóides presentes no veneno de S. saevissima e compará- los com aqueles de outras espécies de formigas lava-pés estudadas. • Determinar a natureza das proteínas de veneno da espécie S. saevissima, e compará-las com o que se conhece de outras espécies. 14 Figura 1. Exemplo de um formigueiro de lava-pés, seccionado ao meio para exibir a estrutura interna de túneis. Os corpúsculos brancos são os estágios imaturos das formigas. Figura 2. Imagem de um ninho de lava-pés sendo criado em laboratório 15 Figura 3. Micrografia eletrônica das peças bucais de uma operária maior de Solenopsis saevissima, evidenciando características da espécie, como costuras completas da mandíbula e grau de desenvolvimento do dente mediano do clípeo (centro da imagem). Figura 4. Extrato purificado de alcalóides de veneno extraído a partir de três formigueiros de Solenopsis saevissima 16 Referências BAER, H.; LIU, T.Y.; ANDERSON, M.C.; BLUM, M.; SCHMIDT, W.H.; JAMES, F.J. protein components of the fire ant venom (Solenopsis wagneri). Toxicon, v. 17, p. 397-405, 1979. BILLEN, J.; ITO, F.; TSUJI, K.; SCHOETERS, E.; MAILE, R.; MORGAN, D. Structure and chemistry of the Dufour gland in Pristomyrmex ants (Hymenoptera, Formicidae). Acta Zoologica, v. 81, p. 159-166, 2000. BLUM, M. S.; WALKER, J. R.; CALLAHAN, P. S.; NOVAK, A. F. Chemical, insecticidal, and antibiotic properties of fire ant venom. Science, v. 128, p. 306- 307, 1958. BLUM, M. S.; ROBERTS, J. R.; NOVAK, A. F. 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T.; KUNZMANN, N.; VANDERWOUDE, C.; SANETRA, M.; CROZIER, R. H. Population genetics and history of the introduced fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), in Australia. Australian Journal of Entomology, v. 44, p. 37-44, 2005. HOFFMAN, D. R.; DOVE, D. E.; JACOBSON, R. S. Allergens in Hymenoptera venom XX. Isolation of four allergens from imported fire ant (Solenopsis wagneri) venom. Journal of Allergy and Clinical Immunology, v. 82, p. 818-827, 1988. HOFFMAN, D. R. Allergens in Hymenoptera venom XXIV.: The amino acid sequences of imported fire ant allergens Sol i II, Sol i III and Sol i IV. Journal of Allergy and Clinical Immunology, v. 91, p. 71-78, 1993a. 18 HOFFMAN, D. R. Allergens in Hymenoptera venom XXV. The amino acid sequences of antigen 5 molecules and structural basis of antigenic cross-reactivity. Journal of Allergy and Clinical Immunology, v. 92, p.707-716, 1993b. HOFFMAN, D. R. Fire ant allergy. Allergy, v. 50, p. 535-544, 1995. HOWELL, G.; BUTLER, J.; DESHAZO, R.D.; FARLEY, J. M.; LIU, H. L.; NANAYAKKARA, N. P. D.; YATES, A.; YI, G. B.; ROCKHOLD, R. W. Cardiodepressant and neurologic actions of Solenopsis invicta (imported fire ant) venom alkaloids. Annals of Allergy and Asthma Immunology, v. 94, p. 380- 386, 2005. JONES, T. H.; BLUM, M. S. Ant venom alkaloids from Solenopsis and Monomorium species. Tetrahedron, v. 38, p.1949-1958, 1982. JUNG, R. C.; DERBES, V. J.; BURCH, A. D. Skin response to solenamine, a hemoloytic component of fire-ant venom. Dermatologica Tropica, v. 2, p. 241- 244, 1963. LECLERCQ, S.; THIRIONET, I.; BROEDERS, F.; DALOZE, D.; VAN DER MEER, R.; BRAEKMAN, J. C. Absolute configuration of the solenopsins, venom alkaloids of the fire ants. Tetrahedron, v. 50, p. 8465-8478, 1994. LOFGREN, C. S.; BANKS, W. A.; GLANCEY, B. M. Biology and control of imported fire ants. Annual Reviews in Entomology, v. 20, p. 1-30, 1975. LUNZ, A. M.; HARADA, A. Y.; AGUIARI, T. S., CARDOSO, A. S. 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CAPÍTULO 1 Uma lista preliminar dos inquilinos encontrados dentro de ninhos de formigas lava-pés no Sudeste Brasileiro 22 A preliminary account on the inquilines of fire ant mounds of Southeastern Brazil Solenopsis Westwood (Hymenoptera: Formicidae) is a large, cosmopolitan genus of myrmicine ants with about 277 species (Bolton, 2006). Twenty Solenopsis species of the Americas have unusually large polymorphic workers and were baptized “fire ants” after their aggressive behavior and painful stings. Fire ants build earthen nests directly on the soil, which may take years to reach maturity (TSCHINKEL, 2006). Some of these nests can attain considerable dimensions over time, and nests as big as 40cm high and over 100cm of base diameter have been observed in Brazil (DALL’AGLIO-HOLVORCEM, 2006; authors’ personal observations). Their internal structure is a labyrinth of honeycomb-like interconnecting tunnels that can provide shelter and a protected environment for the ants and their brood as well as to other arthropod inquilines. Remains of prey, litter, and even the brood and stray or sick ants can serve as food for these inquilines, which are many times tolerated or left unnoticed by the inhabiting ants. There is ample available literature on the association of ants and their inquilines (e.g. AKRE; RETTENMEYER, 1966; DAVEY, 1945; DONISTHORPE, 1927; WHEELER, 1960), and some investigations on the inquilines of fire ants have been carried out (e.g. COLLINS & MARKIN, 1971; BRUCH, 1926; HAYS, 1958; HERMANN et al., 1970). Curiously, no direct investigation of inquilines inside fire ant mounds was yet performed in Brazil. Thus the main goal of this study is to compile a preliminary list of arthropod inquilines associated with fire ant mounds found during field inspections in two different regions of Southeastern Brazil. Materials and Methods Collections were made at two distinct areas over the year of 2007: 1) in the university campus of Sao Paulo State University of Rio Claro, Sao Paulo State, and 2) in a house garden in the municipality of Pedro do Rio, Rio de Janeiro State. Both regions are located at about 600-700 m above sea level, with local temperatures varying 10-30°C over the year, and annual relative humidity around 40-70%. However, the sites are nearly 1,000km apart and differ in terms of local soil and vegetation – first site was dominated by grassland fields whilst second was pastures. 23 The inquilines were directly collected from the fire ant mounds by extracting gradually deeper small portions from the nests with a spade and visually inspecting these inside a plastic tray rimmed with Teflon paint. This way the ants were unable to leave the tray while we searched for other arthropods within the trays. Specimens were always killed and preserved in alcohol 80%, being later sent for identification by specialists. If inquilines were brought to the laboratory along with great portions of the original host fire ant nests, an attempt was made to rear them with the ants inside artificial colonies kept inside plastic trays rimmed with Teflon paint. Results and Discussion Over 20 fire ant nests were inspected at Rio Claro, while 11 nests were inspected at Pedro do Rio. It is worth stressing that no nests of S. saevissima were found in São Paulo, while no nests of S. invicta were found in Rio de Janeiro, illustrating how each species, although morphologically similar, is adapted to the local abiotic conditions. The biological reasons driving the geographic distribution of fire ants are still not fully understood (ROSS; SHOEMAKER, 2005), and the general biology of S. saevissima (including habitat requirements) is poorly known. About 23 species of arthropod inquilines were collected, presented in Table 1. Some of the most commonly found species are discussed further below. Coleoptera – By far, the tenebrionid Blapstinus cf. punctualus was the most frequently found inquiline among nests of S. invicta, occurring in around 50% of the inspected nests, however at low numbers of 2-4 individuals at the topmost inspected area of the nests. We strongly suspect the one tenebrionid larva found belongs to this species, but we cannot be certain as yet. It was simply ignored by the ants while inside the tray, while moving around rather slowly and suddenly stopping at times, probably to avoid attracting too much attention. Ataenuis sp. were found in about 15% of the nests in Sao Paulo. It could freely move amongst the ants and was never attacked, even when running about. Specimens of this very genus were also found by COLLINS; MARKIN (1971) in mounds of S. invicta in the US, but they were suspected to be incidental intrusions as they were found in small numbers. Yet these authors never reported having observed how Ataenuis beetles interacted with the ants. All collected specimens of Throcidae were obtained from only one nest of S. saevissima of particularly large proportions (over 100 cm wide and 40 cm high). 24 Rover beetles of the genus Myrmecosaurus are common inhabitants of fire ant mounds in Brazil, Argentina and in the US, probably having been introduced in the latter country together with S. invicta (SEEVERS, 1965; COLLINS; MARKIN, 1971). Apparently, most myrmecophilous beetles so far remain undetected by the ants from obtaining their cuticular hydrocarbons (VANDER MEER; WOJCIK, 1982; WOJCIK, 1990). Thysanura - Allotrichotriura saevissima was found in 5 nests of S. saevissima, with some of the collected specimens being used for species description elsewhere (MENDES et al., 2009). About 3-4 specimens were found in deeper areas of each analyzed mound. They were fast-moving and difficult to collect. Some specimens were successfully brought to the laboratory with a large portion of their original host colony, where they were reared for over a week. Inside the artificial colonies, the thysanurans remained lingering at the litter piles, where they were apparently feeding upon freshly-deposited debris. They were completely ignored by the ants, but avoided prolonged contact by rapidly moving around. An “apparently undescribed species” of Nicoletiidae was frequently found within nests of S. invicta in the US (see COLLINS; MARKIN, 1971), and this could well correspond to the same species based on the author’s notes. However, they were unable to rear the insects in the laboratory and thus report behavioral observations. Acari – An unidentified scale-like species of Johnstonianidae was found over eggs and brood of two colonies of S. invicta, at large numbers (>100). It seems likely that they were feeding upon this brood and thus were parasitic in these colonies, what might hold some potential as a biocontrol agent. This occurrence would thus merit further investigation. Hemiptera – An apparently undescribed species of Dallasiela (Dallasiela) sp. was found at the number of 1-6 individuals at the topmost regions of 6 nests of S. invicta. The occurrence of burrower (Cydnidae) bugs inside fire ant mounds is unprecedented, and very little is known about the general biology of these insects. The specimens observed moved freely among the ants and were left unnoticed. Anisotermitinae – Over 50 specimens of an undescribed species of Anisotermitinae were found in one mound of S. invicta and one mound of S. saevissima, comprising both mature and young forms (even eggs) distributed in a uniform pattern within the nests; two reproductive nymphs were collected on one occasion. One of the nests was revisited two times over a period of three months, 25 and still contained the termites within. Records of the occurrence of termites inside ant mounds are rare in the literature (CRIST; FRIESE, 1994; SHELTON et al., 1999; DIEHL et al., 2005) and practically none is known about the reasons underlying these associations. Attempts to bring and rear the termites within the ants in the laboratory proved fruitless, as the fragile termites died and dehydrated in a matter of minutes after being moved from the nests. Diptera – Wingless puliciphorans were often observed frantically running among the ants, and were quite difficult to spot and collect. Wingless scuttle flies were already reported in previous inspections of fire ant nests (see WOJCIK, 1990 and references therein). Their diminutive size and rapid movements may have rendered them undetected by most researchers. Pseudacteon are parasitic flies that attack fire ant workers, apparently being attracted by the alarm pheromones and alkaloids (CHEN; HENRY, 2009) released during the exposure of the mound interiors. The presented list briefly illustrates the gap of knowledge about Brazilian inquilines of ant mounds. Many of the collected species are yet undescribed and all belong to biological groups whose biology is basically unknown. The fact that most inquilines found (except for some coleopterans and termites) lacked immature forms within the inspected mounds would be indicative that they only occur in the mounds as adults. We think that some naturally-occurring soil inhabiting species would be seeking protection against predators and / or abiotic alterations. Those species which had immature forms may very well be completing their life cycles within the ant nests. As future perspectives, we are currently trying to obtain additional specimens and working in describing the new taxa. Some of the most frequently found specimens are being investigated as to obtain information about the nature of their association with fire ants. 26 Table 1. Inquiline arthropods collected from fire ant nests in Southeastern Brazil. Inquiline arthropod N. collected Host fire ant species (no. nests where found) Arachnida Salticidae Castianeira sp. Theridiidae Euryopis sp. Coleossoma sp. 1 1 1 S. invicta (1) S. invicta (1) S. saevissima (1) Acarina Johnstonianidae >100 S. invicta (3) Insecta Hemiptera Cydnidae Dallasiellus (Dallasielus) sp. 7 S. invicta (4) Thysanura Nicoletiidae Allotrichotriura saevissima sp.nov. 8 S. saevissima (5) Diptera Phoridae Puliciphora sp. nov. Pseudacteon tridens 5 10 S. richteri (1) / S. saevissima (5) S. invicta (3) / S. saevissima (4) Hymenoptera Formicidae Pheidole sp. Labauchena daguerrei 9 >30 S. invicta (1) S. invicta (1) Isoptera Termitidae Apicotermitinae gen. nov. / sp. nov. >100 S. invicta / S. saevissima (2) Coccoidea Pseudococcidae Dysmicoccus sp Pseudococcus sp. Planococcus sp. >30 >30 >20 S. invicta (1) S. saevissima (1) S. saevissima (1) Coleoptera Endomychidae (larvae) Tenebrionidae Blapstinus cf. punctulatus Larva Scarabaeidae Ataenius elongatus Carabidae (larva) Staphylinidae Throcidae (Coleoptera) 3 15 1 11 1 >30 >50 S. saevissima (1) S. invicta (6) S. invicta (1) S. invicta (5) S. invicta (1) S. saevissima (1) S. saevissima (1) Collembola Entomobrya nivalis Lepidocyrtus sp. nov. Seira sp. nov. 1 Seira sp. nov. 2 20 6 3 4 S. invicta (2) S. invicta (1) S. invicta (1) S. invicta (1) 27 References: AKRE, R. D.; RETTENMEYER, C. W. Behaviour of Staphylinidae associated with army ants (Formicidae: Ecitonini). Journal of the Kansas Entomological Society, v. 39, p. 747-782, 1966. BRUCH, C. Orugas mirmecofilos de Hamearis epulus signatus Stichel. In: Wheeler, W. M. The Social Insects. Kregan Paul, Trench, Trubner & Co. Ltd. London, 378 pp, 1926. CHEN, K. R. S.; HENRY, Y. F. Fire ant venom alkaloids act as key attractants for the parasitic phorid fly, Pseudacteon tricuspis (Diptera: Phoridae). Naturwissenschaften, v. 96, p. 1421-1429, 2009. COLLINS, H. L.; MARKIN, G. P. Inquilines and other arthropods collected from nests of the imported fire ant, Solenopsis saevissima richteri. Annals of the Entomological Society of America, v. 64, p. 1376-1380, 1971. CRIST, T. O.; FRIESE, C. F. The use of ant nests by subterranean termites in two semiarid ecosystems. The American Midland Naturalist, v. 131, p. 370-373, 1994. DALL’AGLIO-HOLVORCEM, C. G. Estudos populacionais e taxonômicos de formigas lava-pés, Solenopsis invicta (Hymenoptera: Formicidae), e da fenologia de seus parasitóides do gênero Pseudacteon (Diptera: Phoridae). Doctoral dissertation, IB / UNICAMP, 2006. DAVEY, H. W. Parasites of ants. Victorian Naturalist, v. 62, p. 105, 1945. DIEHL, E.; JUNQUEIRA, L. K.; BERTI-FILHO, E. Ant and termite mound coinhabitants in the wetlands of Santo Antonio da Patrulha, Rio Grande do Sul, Brazil. Brazilian Journal of Biology, v. 65, n. 3, p. 431-437, 2005. DONISTHORPE, H. The guests of British ants, United Kingdom Press. 1927. 28 HAYS, S. B. The present status of the imported fire ant in Argentina. Journal of Economical Entomology, v. 51, p. 111-112, 1958. HERMANN, H. R.; BLUM, M. S.; HUNT, A. N. Myrmecophilous arthropods associated with the imported fire ant, Solenopsis saevissima (Hymenoptera: Formicidae). Proceedings of the London Academy of Science, v. 33, p. 13-18, 1971. MENDES, L.; FOX, E. G. P.; SOLIS, D. R.; BUENO, O. C. New Nicoletiidae (ZYGENTOMA: INSECTA) from Brazil living in fire ant nests. Papéis Avulsos de Zoologia, v. 49, p. 467-475, 2009. ROSS, K. G.; SHOEMAKER, D. D. Species delimitation in native South American fire ants. Molecular Ecology, v. 14, p. 3419-3438, 2005. SEEVERS, C. H. The systematics, evolution and zoogeography of staphylinid beetles associated with army ants. Fieldiana, v. 47, p. 138-351, 1965. SHELTON, T. G.; VOGT, J. T.; APPEL, A. G. ; OI, F. M. Observations of Reticulitermes spp. in Solenopsis invicta mounds (Isoptera: Rhinotermitidae, Hymenoptera: Formicidae). Sociobiology, v. 33, p. 265-275, 1999. TSCHINKEL, W. R. The Fire Ants. Harvard University Press, Cambridge, 2006. WHEELER, W. M. Ants, their structures, development and behavior. 3rd ed. Columbia University Press, New York, 1960. WOJCIK, D. P. Behavioral interactions of fire ants and their parasites, predators and inquilines. Pages 329-344. in VANDER MEER, R. K., JAFFE, K., CEDENO, A., Editors. Myrmecology: A World Perspective, Studies in Insect Biology. Westview Press, San Francisco, pp. 329–344, 1990. VANDER MEER, R. K.; WOJCIK, D. P. Chemical mimicry in the myrmecophilous beetle Myrmecaphodius excavaticollis. Science, v. 218, p. 806-808, 1982. CAPÍTULO 2 SOBRE UM NOVO NICOLETIIDAE (ZYGENTOMA: INSECTA) DO BRASIL VIVENDO COM FORMIGAS LAVA-PÉS (HYMENOPTERA: FORMICIDAE) Papéis Avulsos de Zoologia: Volume 49(34):467�475, 2009 Desenhos de Luis. F. Mendes 30 NEW NICOLETIIDAE (ZYGENTOMA: INSECTA) FROM BRAZIL, LIVING IN FIRE ANT (HYMENOPTERA: INSECTA) NESTS Abstract A new Nicoletiidae (Subnicoletiinae) myrmecophilous silverfish (Zygentoma) is described from Rio de Janeiro, Brazil, found living with in a fire ant (Solenopsis saevissima, Formicidae: Myrmicinae) nest: Allotrichotriura saevissima gen. nov. sp. nov. is compared with other genera and subgenera known in the subfamily. The main diagnostic features would include the combination: body shape, body and head setae, morphology of praetarsus, and number of abdominal stylets and vesicles. Although further quests were attempted at the type-locality, only the original described material, exclusively composed of females, remains know. 31 Introduction The fauna of Nicoletiidae (Zygentoma) in Brazil remains largely unknown and integrates currently 19 known species distributed in 11 genera, including leaf-litter, soil-dwelling (edaphic: ED), myrmecophilous (MY), termitophilous (TE - all living with Termitidae) species and species living with yet undetermined hosts (UH), or even in unknown biotopes (UB), as well as cave-dwellers (troglobites: TR). All known subfamilies of Nicoletiidae occur in that country, being Atelurinae (13 species), the most diverse group. Grassiella (Atelurinae) is so far the most diverse genus, with six known Brazilian species, of which five are endemic. One new species solely represented by female specimens belonging to a new genus of Subnicoletiinae was obtained from a fire ant (Solenopsis saevissima, Formicidae: Myrmicinae) nest from Rio de Janeiro State. It is described below and the new genus is compared with the known genera and subgenera in that subfamily. Brazilian nicoletiidae were reported from Amazonas (AM), Bahia (BA), Espírito Santo (ES), Goiás (GO), Mato Grosso (MT), Minas Gerais (MG), Pará (PA), Pernambuco (PE), Rio de Janeiro (RJ), Santa Catarina (SC), and São Paulo (SP), according with the following alphabetic list. Authors of the irrespective citations are reported; species known as endemic to Brazil are marked with an *. Subfamily ATELURINAE: *Atelurina pernambucensis WYGODZINSKY, 1943 - PE (UH) (Wygodzinsky, 1943a) *Goiasatelura goianella WYGODZINSKY, 1942 - GO (TE) (Wygodzinsky, 1942) *Goiasatelura goianensis WYGODZINSKY, 1942 - GO (TE - Syntermes, Nasutitermitinae) (Wygodzinsky, 1942) *Grassiella aepsera WYGODZINSKY, 1958 - RJ (MY - Camponotus, Formicinae, and Atta, Myrmicinae; eventually TE also) (WYGODZINSKY, 1958a) *Grassiella amazonica Mendes, 1996 - AM (UB) (MENDES, 1996) *Grassiella artipoda Wygodzinsky, 1958 - ES (UB) (WYGODZINSKY, 1958a) *Grassiella carioca Wygodzinsky, 1958 - RJ (UB) (WYGODZINSKY, 1958a) *Grassiella negroensis Mendes, 2002 - AM (MY - undetermined Myrmicinae) (MENDES, 2002) Grassiella praestans Silvestri, 1898 - MG SC, SP, RJ (MY – unidentified ants) (ESCHERICH, 1905 sub Atelura, SILVESTRI, 1946, WYGODZINSKY, 1958a) 32 *Heterolepidella synoeketa (SILVESTRI, 1901) - MT (TE - Eutermes debilis, Nasutitermitinae) (ESCHERICH, 1905 sub Atelura; SILVESTRI, 1901a,c, 1903 sub Grassiella) *Heterolepidella termitobia (SILVESTRI, 1901) - MT(TE - Anoplotermes tenebrosus and Amitermes amifer, Amitermitinae) (ESCHERICH, 1905 sub Atelura; SILVESTRI, 1901a,c, 1903 sub Grassiella) Lasiotheus nanus (ESCHERICH, 1903) - RJ (MY - Prenolepis, Formicinae) (WYGODZINSKY, 1958a, wrongly identified as Cryptocephalina minutella, rectified by MENDES, 1986) *Pseudogastrotheus synterminus (SILVESTRI, 1946) - RJ (MY - undetermined ants; and TE - Syntermes, Nasutitermitinae) (SILVESTRI, 1946, WYGODZINSKY, 1958a, both sub Gastrotheus) Subfamily COLETINIINAE: *Coletinia brasiliensis MENDES & FERREIRA, 2002 - BA (TB in the “Toca do Morrinho” Cave) (MENDES & FERREIRA, 2002) Subfamily CUBACUBANINAE: * Anelpistina spelaea (GALÁN, 2001) - BA (TB in the “Toca da Boavista” Cave) (Galán, 2001 sub Cubacubana) Subfamily NICOLETIINAE: Nicoletia phytophila Gervais, 1844 (females only) - PA (ED) (PICCHI, 1972 as N. meinerti). SILVESTRI (1912) suggested N. meinerti as a synonym for N. phytophila, and WYGODZINSKY (1980) (no precise data, eventually the Picchi’ material from Pará) registered the presence of N. phytophila in the Brazilian Amazon, confirming Silvestri’s synonymic proposal. Also present in the rain forests of AM (unpublished data). Subfamily SUBNICOLETIINAE: *(?) Hematelura convivens ESCHERICH, 1906 - PA (TE - undetermined termites) (ESCHERICH, 1906). Species described from a female, and the only one holotype specimen is almost certainly lost; incomplete description lacking details puts the validity of this species in question. Trichatelura borgmeieri SILVESTRI, 1933 - GO (MY – army ants: Eciton crassicorne, E. diana, E. dulcis, E. minense, E. praedator and E. sclechtendali, Dorylinae) (WYGODZINSKY, 1943b) 33 Trichatelura manni (CAUDELL, 1925) - GO (MY - army-ants: Eciton crassicorne and E. praedator, Dorylinae) (WYGODZINSKY, 1943b) Note 1: The validity of Nicoletia neotropicalis Silvestri, 1901 - MT (ED) (SILVESTRI, 1901b,c; ESCHERICH, 1905) warrants investigation; the conspecificity of samples from Argentina, Brazil, Paraguay and Uruguay recorded under this name needs to be revisited (they all hardly pertain Nicoletia, and they may not even belong to Nicoletiinae). Note 2: Nicoletia armata SILVESTRI, 1901 (ED), eventually a Cubacubaninae in need of revision, was reported by ESCHERICH (1905) to occur in Brazil: “…Silvestri fand sie in Brazilien, Uruguay und Paraguay…”; as a matter of fact, this enigmatic species was registered by Silvestri (1901b,c) from Argentina, Paraguay (Paraná) and Uruguay, but never from Brazil. Material and Methods The studied material is deposited in the entomological collections of Museu de Zoologia da Universidade de São Paulo, SP, Brazil (MZUSP) and Zoologia of the IICT / JBT, Lisbon, Portugal (CZ - former Centro de Zoologia). Allotrichotriura were dissected under a stereomicroscope, being the dissected pieces mounted from ca. 70-80 % ethanol directly in ‘Tendeiro’ liquid, and dried at 40ºC for about one week (before observation) and for 2-3 weeks (before storage, until solidification); whole specimens were also preserved in alcohol. Observations and species identification were performed with a compound microscope and drawings made with a camera lucida. Results and Discussion Allotrichotriura gen. nov. Description: Female: Nicoletiidae Subnicoletiinae of small body size (< 4 mm), ateluriform (short and stout), lacking pigmentation and without scales, most of the setae thin and very short (only a few acute or apically slightly bifurcated macrochaetae on the head and tergites). Head exposed, setose. Nota and abdominal tergites and sternites, with the setae arranged in several irregular rows. Incisive and molar areas of mandibles well developed. Galea and lacinia equally developed; galea with 1 apical conule only, the prostheca not clearly longer than the apical tooth of lacinia. Maxillary and labial palps typical. Praetarsus simples and complete. All the 34 abdominal segments exposed. Stylets on abdominal segments VI-IX (4 pairs), the vesicular structures reduced to the pseudovesicles VII. Subgenital plate widely elliptical, the ovipositor spindle-shaped, with thin setae only and clearly longer than level of stylets IX. Cerci and paracercum short, lacking spines. Male unknown. Type-species: Allotrichotriura saevissima sp. nov. Etymology: From the Greek, Allos: other, and from Trichotriura Silvestri, 1918, one West African genus eventually close to the new endemic Brazilian genus. Discussion: The new genus fits in Subnicoletiinae (sensu MENDES, 1994), probably a polyphiletic group as judiciously suggested by Smith (1998) known in the Neotropical, Afrotropical, Oriental and Australian Regions. Following genera are included, namely Hematelura Escherich, 1906, Hemitrinemura Mendes, 1994, Metrinura Mendes, 1994, Subnicoletia Silvestri, 1908, Subtrinemura Smith, 1998, Trichatelura Silvestri, 1932, Trichotriura Silvestri, 1918, Trichotriurella Mendes, 2002, Trichotriuroides Mendes et al., 1994, Trinemura Silvestri, 1908 and Trinemurodes Silvestri, 1916. All the genera belonging to Trinemura s. l. (SILVESTRI, 1908, MENDES, 1994, Smith, 1998 – so, Trinemura s. s., Hemitrinemura, Metrinura and Subtrinemura) are immediately discernible from Allotrichotriura gen. nov. due to the number of abdominal stylets and the larger subgenital plate, being Trinemura s. s. even more distinct for presenting more numerous abdominal vesicles. The same can be stated relatively to Trinemurodes Silvestri, 1916 that lacks, furthermore, a praetarsal empodium. Subnicoletia Silvestri, 1908 presents, like the preceding ones, more numerous abdominal stylets (IV-IX) and vesicular structures (IV-VII). Besides, in all these genera the specimens are typically “nicoletiid-shaped”, with long thin and parallel-side bodies. Hematelura (ESCHERICH, 1906; WYGODZINSKY, 1958b) and mainly Trichatelura Silvestri, 1932, Trichotriura Silvestri, 1918, Trichotriurella Mendes, 2002 and Trichotriuroides Mendes et al., 1994 have, like the new genus, more or less “atelurid- shaped” bodies, round, short and broad, as well as a clear reduction of both, the number of abdominal stylets and of vesicular structures; the last aforementioned four genera share with Allotrichotriura the single apical conule in the galea but they have stylets restricted to the urosternites VII-IX (3 pairs only) or these structures can be even less numerous (one pair only in Trichotriurella). Furthermore: 35 Trichatelura, ecitophilous and Neotropical, with 2 known species from Brazil, as reported, has a single row of strong setae along the posterior border of the urotergites, thin and cylindrical labial palp apical article, very different subgenital plate, and much shorter ovipositor; in the new genus all tergal and sternal setae are similarly developed, thin, short and arranged in several irregular rows, being slightly more dense and more developed on posterolateral areas only, and a single macrochaeta does occur. Trichotriura, termitophilous from Nigeria, with even smaller specimens, shows, like the preceding genus, different dorsal setation, being the urotergites provided with one only hind row of well-developed setae; furthermore, the labial palp distal article is also almost sub-cylindrical. Trichotriuroides, monotypical and endemic from the Equatorial Guinean island of Bioko (formerly Macias Nguema, before that Fernando Po) seems more similar to Allotrichotriura though the comparison remains difficult as the new genus type-series includes exclusively females, while Trichotriuroides remains known from one only male. Main differences seem to concern the almost completely concealed abdominal tergite I due to the proportional development of the thorax (free in the new genus), the cylindrical labial palp distal article (round in Allotrichotriura), the distinct empodium, the setae density along the body (mainly nota) and the lack of thoracic macrochaetae. Trichotriurella, from the former Zaire and also monotypical, with mature specimens also smaller than those of the new genus, is similarly known from females only; among other dissimilarities, there is different cephalic setation, very distinct mandibles and maxillae, longer antennae and only one pair of abdominal stylets. Hematelura, from Africa with one only representative (autochthon?) in Brazil, shows (at least in the Afrotropical species we could study) two well developed conules on the galea. This genus presents some variability in the number of abdominal stylets and vesicles, and the 3 known species that completely lack scales, H. convivens Escherich, 1906, H. setosa (SILVESTRI, 1918 sub Monachtinella) and H. delamarae Wygodzinsky, 1958 are quite distinct from Allotrichotriura. H. convivens, from Brazil, if congeneric with the remaining species and if correctly characterized, has vesicular structures on the segments VI-VII opposite to all the remaining Hematelura and to the condition in Allotrichitriura gen. nov.; furthermore, the ovipositor is much longer than of the new genus. H. setosa, known exclusively from type material from Guinea, 36 with 5 pairs of stylets (V-IX), is the only species to present (in males) a conspicuous projection on the antennal pedicellus; as a rule in the known females, the ovipositor is much longer than in the new genus; at last H. delamarei, from the Ivory Coast, known only by its 5 mm long holotype male, also with 5 pairs of abdominal stylets, shows a distinct, acicular empodium and peculiar, scattered, delicate, lanceolate setae on the urotergites (nothing similar occurs in the new genus). Allotrichatelura saevissima sp. nov. (Figs. 1-20) Type-material: Holotype female, BRAZIL, Rio de Janeiro: Pedro do Rio, 22º 20’32.64 S, 43º 7’58.96 W, 730 m altitude, 8/5/2006, within a fire ant (Solenopsis saevissima) nest, coll. E.G.P. Fox, (CEIS/UNESP). Paratypes: Same data as holotype, 1 female (MZUSP), 1 female (CZ- 5276). Description: Female: Body length: 3-3.2 mm; thorax length: 1.4 mm; thorax width: 1.4 mm; maximum length of antennae: maximum measured of 1.3 mm; cerci length: 0.9 mm; terminal filament short, always damaged. Hypodermal pigmentation absent, the setae and macrochaetae hyaline. Head (Fig. 1) wider than long, the cephalic capsule with numerous thin short setae and with a few frontal acute macrochaetae. Antennae short, without peculiar features. Incisive and molar areas of mandible well developed (Fig. 2). Maxillae without especial characteristics the prostheca slightly longer than the apical tooth of lacinia, as long as the galea, this one with one only short apical conule (Fig. 3). Maxillary palp delicate, the distal article cylindrical and longer than the previous one, and with several apical sensilla (Figs. 4, 5). Labium as usual, labial palp (Fig. 6) medium-size, its distal article ovoid, ca. 1.2 times longer than wide and with the six typical apical papillae. Nota short and wide, with numerous irregular rows of minute thin setae, their posterior border almost straight (pronotum) to slightly depressed (metanotum); only one very short, apically bifid macrochaetae, stronger though not longer than the usual setae, occurs on the anterior-lateral angle of pronotum (Fig. 7). Legs without especial features, the tibias (Figs. 8, 9) ca. 3 times longer than wide, the empodium simple and complete (Fig. 10). Urotergites I-VIII as the nota, with several thin short setae, more numerous on the infralateral area; one only stout macrochaeta present (Fig. 11), its robustness 37 increasing from the anterior to the posterior segments; infralateral areas of urotergite IX poorly dilated, as in Fig. 12. Urotergite X sub-trapezoidal (Fig. 13), much shorter than wide at base, its posterior notch obtuse, not especially depressed; 1+1 infralateral plus 1+1 shorter lateral macrochaetae on the posterior border and some rare discal thin setae. Urosternite I almost glabrous with rare submedian setae, the II with 1+1 lateral plus 1 median well delimited groups of setae (Fig. 14); abdominal sternites III-VII with abundant thin small setae, uniformly distributed, like in the dorsal plates (Fig. 15). Four pairs of abdominal stylets, on segments VI-IX (Fig. 16); only the pseudovesicles VII present. Posterior border of urosternite VII clearly concave, the subgenital plate wide and short, parabolic to almost triangular (Fig. 17). Coxites VIII and IX typical (Fig. 18), the ovipositor spindle-shaped and clearly exceeding the level of the stylets IX apex; gonapophyses VIII and IX as in Figs. 19, 20 with ca. 6 divisions. Terminal filaments short, without special features. Male unknown. Etymology: The new species was baptized after its fire ant host-species, Solenopsis saevissima. References ESCHERICH, K. Das System der Lepismatiden. Zoologica, v. 43, p. 1-164, 1905. ESCHERICH, K. Beiträge zur Kenntnis der Thysanuren. II Reihe. Zoologischer Anzeiger, v. 30, p. 737-749, 1906. GALÁN, C. Nueva especie cavernícola de Thysanura Nicoletiidae de la Toca da Boavista (Estado de Bahía, Brasil). Boletín de la Sociedad Venezoelana de Espeleologia, v. 35, p. 13-19, 2001. MENDES, L. F. Nova contribuição para o conhecimento dos tisanuros africanos (Zygentoma: Lepismatidae e Ateluridae). Revue de Zoologie Africaine, v. 100, p. 213-227, 1986. MENDES, L. F. Evolutionary relationships among the Nicoletiidae (Insecta, Zygentoma). Acta Zoologica Fennica, v. 195, p. 98-103, 1994. 38 MENDES, L. F. Novos dados e descrições de tisanuros (Microcoryphia e Zygentoma: Insecta) da América do Sul. Garcia de Orta, v. 21, p. 129-144, 1996. MENDES, L. F. Novos dados sobre tisanuros (Microcoryphia e Zygentoma: Apterygota) e descrição de uma nova espécie do Brasil. Garcia de Orta, v. 24, p. 81-87, 2002. MENDES, L. F.; FERREIRA, R. L. On a new cave-dwelling Nicoletiidae (Zygentoma: Insecta) from Brazil. Garcia de Orta, v. 24, p. 101-106, 2002. PICCHI, V. D. Parthenogenetic reproduction in the silverfish Nicoletia meinerti (Thysanura). Journal of the New York Entomological Society, v. 80, p. 2-4, 1972. SILVESTRI, F. Descrizioni di nuovi termitofili e relazioni con gli ospiti. IV. Thysanura. Bolletino del Museo de Zoologia e Anatomia Comparata di Torino, v. 16, p. 13- 15, 1901a. SILVESTRI, F. Materiali per lo studio dei Tisanuri. III. Nuove specie di Nicoletia. Bolletino della Societá Entomologica Italiana, v. 33, p. 223-227, 1901b. SILVESTRI, F. Materiali per lo studio dei Tisanuri.V. Tisanuri trovate da altre e da me nell’America Meridionale. Bolletino della Societá Entomologica Italiana, v. 33, p. 229-247, 1901c. SILVESTRI, F. Contribuzione alla conoscenza dei termitidi e termitofili dell’America Meridionale. Termitofili (III – Thysanura). Redia, v. 1, p. 179-181, 1903. SILVESTRI, F. Thysanura. In: MICHAELSEN, W. & HARTMEYER, R. (eds.). Die Fauna Südwest-Australiens. Ergebnisse der Hamburger Südwest- australischen Forschungsreise 1905, Gustav Fischer, Jena, 1908. 39 SILVESTRI, F. Tisanuri finora noti del Messico. Bolletino del Laboratorio de Zoologia Generale e Agraria di Portici, v. 6, p. 204-221, 1912. SILVESTRI, F. Primo contributo alla conoscenza dei termitofili viventi com specie di Syntermes. Commentationes Pontificia Academia Scientiarum, v. 9, p. 515-559, 1946. SMITH, G. Review of the Australian Nicoletiinae (Zygentoma: Nicoletiidae). Invertebrate Taxonomy, v. 12, p. 135-189, 1998. WYGODZINSKY, P. Um novo género e duas novas espécies de lepismatídeo termitófilo do planalto central do Brasil (Lepismatidae, Thysanura). Revista de Entomologia, v. 13, p. 354-359, 1942. WYGODZINSKY, P. Sobre um novo género e uma nova espécie da subfamília «Nicoletiinae» (Lepismatidae, Thysanura) do Estado de Pernambuco (Brasil). Revista Brasileira de Biologia, v. 3, p. 351-353, 1943a. WYGODZINSKY, P. Nota sobre um gênero de lepismatídeo ecitófilo (Thysanura, Lepismatidae). Revista de Entomologia, v. 14, p. 260-262, 1943b. WYGODZINSKY, P. Sobre algunos «Nicoletiidae» americanos (Thysanura, Insecta). Acta Zoológica Lilloana, v. 16, p. 97-120, 1958a. WYGODZINSKY, P. On some Thysanura and Machilida from French West Africa. Bulletin de l’Institut Français de l’Afrique Noire, v. 20, p. 1145-1175, 1958b. WYGODZINSKY, P. A survey of the Nicoletiinae of Europe (Nicoletiidae, Thysanura, Insecta). American Museum Novitates, v. 2695, p. 1-24, 1980. 40 Figures 1-6: Allotrichotriura saevissima gen. nov. sp. nov., female. 1. Head. 2. Mandible. 3. Maxilla. 4. Maxillary palp. 5. Id, detail of the distal article. 6. Labial palp. Scale bars: 0.1 mm. 41 Figures 7-13. Allotrichotriura saevissima gen. nov. sp. nov., female. 7. Antero-lateral area of pronotum. 8. P I. 9. P III. 10. Empodium. 11. Urotergite III. 12. Urotergite IX. 13. Urotergite X. Scale bars: 0.1 mm 42 Figures 13- 17: Allotrichotriura saevissima gen. nov. sp. nov., female. 14. Urosternites I-III. 15. Urosternite V. 16. Urosternite VI. 17. Urosternite VII and subgenital plate. Scale bars: 0.1 mm 43 Figures 18-20: Allotrichotriura saevissima gen. nov. sp. nov., female. 18. Posterior abdomen, ventral (ovipositor outlined). 19. Gonapophyses VIII, distal divisions. 20. Gonapophyses IX, distal divisions. Scale bars: 0.1 mm CAPÍTULO 3 SOBRE AS LARVAS DA FORMIGA LAVA-PÉS Solenopsis saevissima Smith 45 On the morphology of immature stages of the fire ant Solenopsis saevissima (Smith) (Hymenoptera: Formicidae) Abstract Although common in Brazil, the biology of the fire ant Solenopsis saevissima (Smith) is still poorly studied, and fire ants are a specially complicated group. Larval descriptions are useful to genus-level ant systematics. This study presents a detailed description of immatures of all castes of S. saevissima along with scanning electron microscopy imagery. Different larval instars were separated by diagnostic morphological traits which could be confirmed by directly observing moults. Reproductive larvae could be easily identified by their distinctive bodily dimensions and shape. Larvae of S. saevissima proved to be identical to Solenopsis invicta, and mature larvae presented considerable intraspecific variation in some larval characters recently proposed to aid in fire ant species separation (i.e. morphology of head hairs). We now feel that fire ant larval characters may not be useful for species-level identification and phylogeny. 46 Introduction The importance of immature morphology to insect systematics and taxonomy was extensively discussed in previous studies (e.g. FINLAYSON, 1975; WHEELER; WHEELER, 1976; SCHULTZ; MEIER, 1995). The present approach is inserted in a series of studies on ant larvae which attempt to remedy the limitations in the available morphological information on hymenopteran larvae. Solenopsis (Hymenoptera: Formicidae) is a cosmopolitan genus that includes approximately 277 species, of which over 108 occur in the New World (BOLTON, 2006). Some of the largest species are aggressive polymorphic ants trivially known as ‘fire ants’ which are usually distressing in the geographical regions they occur, either as a native or invasive species. The Solenopsis saevissima group of fire ant species (sensu PITTS et al., 2005) includes 13 species of fire ants which are markedly difficult to sort because of the plasticity of morphological characters employed, and because of their strong polymorphism. In an attempt to propose a phylogenetic tree for the species within this complex, PITTS et al. (2005) revisited the morphological characters as originally proposed by TRAGER (1991) and added new ones, including the use of head setae of last-instar larvae. However, there are no morphological descriptions of fire ant larvae currently available in the literature, except for the species S. invicta and S. geminata (WHEELER; WHEELER, 1955; ONEIL; MARKIN, 1975; PETRALIA; VINSON, 1977). The fire ant Solenopsis saevissima Smith is common in Brazil, however still remains a generally poorly studied species, and their larvae were never described. The present study thus aimed at contributing to the body of knowledge about the fire ants by describing each immature stage of S. saevissima with the aid of light and scanning electron microscopy. Methods Obtention of samples. Whole nests of S. saevissima were obtained following the methods of BANKS et al. (1981) at Pouso Alegre (22°13'48''S 45°56'11”W), State of Minas Gerais, and Pedro do Rio (22º20'32”S 43º7'58”W), State of Rio de Janeiro, Brazil. Species identification was made based on TRAGER (1991) and PITTS et al. (2005). The following diagnostic characters were confirmed in our samples: complete mandibular 47 costulae, lack of a medial frontal streak and poorly developed medial clypeal tooth. From three of these colonies, we could obtain immature forms to be used in our descriptions. Additional samples from Ilhéus (14o15´S 39o13´W), State of Bahia, Brazil, were also analysed to confirm the morphological traits and intraspecific variations observed. Voucher specimens of all immature and adult stages of the collected coloines were deposited in the entomological collections of Instituto Biológico and Museu de Zoologia (MZUSP), São Paulo, Brazil. Determination of larval instars. The first larval instar and the last larval instar can be directly identified from hatching larvae and prepupae, and thus be used to bracket others. PETRALIA; VINSON (1979) described characteristics that were unique of each larval instar of S. invicta, and these characteristics were also employed here with S. saevissima. Larval instar characteristics were further validated by observing moulting larvae. Differentiation of larvae from different castes. Worker larvae only differed when mature in bodily dimensions, thus a size interval is given. Gyne and male larvae were considerably larger than worker larvae and presented typical body shapes of their own. These were directly confirmed as they moulted into male or female alate pupae. Description of the immature forms. All collected samples were fixed in Dietrich’s solution (900 ml distilled water, 450 ml 95% ethanol, 150 ml 40% formaldehyde, 30 ml acetic acid) for 24h and then conserved in 70% alcohol. Samples to be analysed under the scanning electron microscope were dehydrated in an alcohol graded series (80-100%; 10-min-dips in each concentration), and critical-point dried (Balzers CPD/030). Dried specimens were then attached to aluminium stubs with double-faced conductive adhesive tape and gold-sputtered with a Balzers SCD/050 sputterer. Observations and images were obtained as soon as possible after sample preparation. Samples to be analysed under the compound microscope were warmed for 15 min in KOH 10% and placed in a small drop of glycerin on a microscope slide. 48 The morphological descriptions were based on over 10 larvae of each instar. The larvae were analyzed and described under a compound light microscope (Zeiss MC80 DX, with maximum magnification of 1000X), and illustrations were obtained with a scanning electron microscope (LEO 435 VP, at 20.0 kV). With a stereomicroscope (Zeiss Stemi SV11, with maximum magnification of 66X) equipped with a micrometric eyepiece, we obtained measures of every stage. All terminology used herein followed Wheeler and Wheeler (1976), and measures, where applicable, are given as mean ± SD followed by the number (n) of individuals analyzed. Further specimens were later mounted on glass slides to rapidly check for intraspecific variations. Comparison with other samples. Last instar larvae of S. invicta from our laboratory and a few specimens of S. saevissima from Bahia were also rapidly analyzed to check for instraspecific variation in the morphological characters proposed by Pitts et al. (2005). Results Egg (Figure 1A): Widely ovoid in shape, about 0.18 mm x 0.25 mm, with the whitish embryo showing through the transparent chorion. No outer ornamentation or orifices. Eclosion occurs through a medial transverse rupture (Figure 1B), apparently as the first instar larva grows beyond the delicate chorion forcing it open. First larval instar (Figure 1C-G): Body profile attoid, 0.31 ± 0.02 mm long x 0.16 ± 0.01 mm wide (n = 5); body length through spiracles 0.52 mm (n = 1) (Figure 1C). There were ten inconspicuous pairs of spiracles, with the first one larger (0.002 mm) than others (0.001 mm). Integument surface smooth, without setation, however with short spines over the posterior abdominal region and around the anus (not shown). Head capsule subelliptical, 0.13 ± 0.02 mm wide (n = 5), without setation and sensilla (Figure 1D). Clypeus and labrum fused to a single semicircular piece (0.035 mm) (Figure 1D); maxillae lobose about 0.02 mm long and 0.02 mm wide; maxillary palps and galea indistinct (Figure 1F). Mandibles transparent and round, bearing two short apical teeth, about 0.025 49 mm long and 0.018 mm wide (Figure 1E). Labium ovoid, about 0.03 mm wide; labial palps indistinguishable (Figure 1G). Second larval instar (Figure 2A-D): Body profile attoid, greatly curved and with anus terminal; 0.48 ± 0.01 mm long and 0.23 ± 0.01mm wide at largest (n = 9); body length through spiracles 0.64 mm (n = 1) (Figure 2A). Body setae scarce and always simple, 0.026-0.030 mm long, concentrated on the dorsal area of the first thoracic somite and over the terminal region of the body (not shown). There are ten pairs of spiracles, with the first slightly larger (0.01 mm) than the rest (0.006 mm) (not shown). Head capsule subelliptical, 0.17 ± 0.01 mm wide (n = 9) (Figure 2B). Head hairs distributed as follows: between six and eight over the occipital border, two or three on vertex, and five on each gena. Antennae difficult to spot and bearing three basiconic sensilla (not shown). Mouthparts: Clypeus fused with labrum to a single short trapezoidal piece about 0.08 mm wide and 0.09 mm long, bearing a row of four simple setae on the fusion line (Figure 1C); there are spiny papillae on the dorsal surface nearest to mouth entrance. Maxillae lobose and 0.049 mm long and 0.05 mm wide, bearing one simple seta at the base (not shown). Mandibles unpigmented and roughly camponotoid in shape, yet with a pronounced apical tooth and a small subapical tooth, measuring 0.05 mm long and 0.033 mm wide at base (Figure 1C). Labium a 0.06mm-wide sphere, with neither palps or spinnerets visible; with dense spines near mouth entrance (not shown). Third larval instar (Figure 3A-C): Body profile roughly dolichoderoid, about 1.22 mm ± 0.01 mm long and 0.48mm ± 0.01 mm wide (n = 172); length through spiracles 1.29 mm (n = 2) (Figure 3A). Body setae uniformly distributed and of three types: deeply bifid (0.02-0.03 mm long), bifid (0.03 mm long) and simple, with curved hook-like tips (0.01-0.05 mm long) (Figure 3A). Simple setae abound all over the body except for the ventral region of the anterior somites (‘food basket’ area), which is naked and without spines. Bifid hairs are also found over most of the body surface, but predominate on the posterior body region. There are ten pairs of spiracles, with the first being slightly larger (0.1 mm) than the rest (0.07 mm) (not shown). Head capsule 0.28 ± 0.01 mm wide (n = 172); subelliptical and presenting three types of hairs: simple with tip hooked (0.04 mm 50 long), smooth and simple (0.007 mm long), and bifid (0.015-0.020 mm long) (not shown). Head hairs distributed as follows: six or seven hairs on the occipital border, with some (1-3) being bifurcated in some specimens, five hook-tipped simple hairs and three or four bifid hairs on the vertex (some specimens had only simple hook- tipped hair), two or three hook-tipped hairs on the frons, between five and eight simple hairs on each gena (some were bifid in some specimens, and one had a 3- branched hair). Antennae slight elevations with three basiconic sensilla (not shown). There was a conspicuous pair of enclosed sensilla at the base of each mandible. Mouthparts (Figure 3B): clypeus and labrum fused in a single trapezoidal piece 0.087mm wide, slightly depressed mesad and with a row of four simple hairs at the intersection; between four and six setaceous sensilla on the anterior face of the labrum and six to seven basiconic sensilla on the posterior face of the labrum, which is densely endowed with spinulose papillae. Maxillae paraboloidal in shape, about 0.05 mm long and 0.037 mm wide, with a hook-tipped hair near the base (some specimens had an additional short simple hair) and two setaceous sensilla; maxillary palpus a simple elevation with four basiconic sensilla, and galea represented by a pair of basiconic sensilla. Mandibles poorly sclerotized, about 0.057 mm long and 0.037 mm wide at base. Labium elliptical, about 0.1 mm wide, and bearing one or two simple setae on the surface below the opening of the sericteries – which is an horizontal slit about 0.04 mm – and a conspicuous cluster of spiny papillae above the sericteries towards the mouth entrance (Figure 3B). Fourth Larval Instar of Worker (Figure 4A-D): Body profile pheidoloid; larvae of different sizes varying within 1.35-2.85 mm long (n = 77) and 0.58-1.3 mm wide (n = 77) (Figure 4A). Dimensions of spiracle peritremes and mandibles of larvae of different sizes always similar. All measurements presented here were taken from a 3.0 mm long larva. Body length through spiracles 4.22 mm. Body setae uniformly distributed and of three types: deeply bifid (0.075 mm), bifid (0.70 mm) and simple (0.055 mm). Simple setae were most common on the ventral region of the anterior somites, while bifid setae were predominant over the rest of the body. Area of ‘food basket’ was naked and without spines (not shown). There are ten pairs of spiracles, the first pair being slightly larger (0.016 mm) than the rest (0.014 mm), and the last pair being smallest (0.100 mm). Head capsule 0.37 ± 0.01 mm wide; subelliptical and with 20-30 setae of two types: simple (0.1-0.12 mm) 51 and bifid (0.57 mm), distributed as follows: seven or eight (rarely nine) hairs on the occipital border, usually bifid (but sometimes central hairs can be simple, as illustrated in Figure 8), two or three hairs on each side of vertex (one of them usually bifid), between two and four simple hairs on the frons, between five and seven simple hairs on each gena (Figure 4B). Antennae clearly visible and with three basiconinc sensilla. There was a pair of enclosed sensilla near the base of the mandibles. Mouthparts: clypeus poorly delimited from the head and rectangular, with four simple hairs along distal border (Figure 4C). Labrum clearly delimited and roughly rectangular, slightly depressed mesad, about 0.12 mm wide and presenting six basiconic sensilla and seven to eight setaceous sensilla on its anterior surface, being densely armed on its ventral surface and borders with rounded and spiny papillae (Figure 4D). Maxilla roughly paraboloidal in shape and measuring 0.085 mm long and 0.047 mm wide, with two setaceous sensilla near the base of the galea. Galea paxiliform and 0.015 mm long, and maxillary palpus digitiform and 0.22 mm long, with the first being tipped with two setaceous sensilla and the latter with four sensilla, being two basiconic, one setaceous and one enclosed. Mandibles ectatommoid in shape, heavily sclerotized and stout (0.1 mm long and 0.037 mm wide) with two apical teeth and two prominent subapical teeth followed by a long blade with two or three molar denticles. Labium rounded, about 0.8mm wide; labial palps being simple elevations about 0.012 mm wide with four basiconic sensilla and on setaceous sensillum on top; labial surface below the palps presenting two or three basiconic sensilla and one or two setaceous sensilla at varied positions; labial surface above the palps endowed with sparse spines directed to the mouth entrance (Figure 4C). Opening of sericteries a horizontal slit about 0.035 mm long with an enclosed sensillum by the end of each extremity. Epipharynx weakly spinulose. Reproductive larvae (Figures 5 and 6): The reproductive larvae differed from worker larvae only on last instar, and by their greater size and altered shape (compare Figures 4A, 5A and 6A). Also, the increment in body size resulted in a decrease of body hair density, thus they look less hairy than worker larvae. Mature larvae (prepupae) of males measured about 4.0 mm, and had a distinct shape with visibly engorged thorax (Figure 6A), and acquired a whitish hue apparently because of a thicker integument. Mature larvae of gynes measured over 5.0 mm long and had greatly swollen abdomens (Figure 5A). 52 A few morphological particularities from worker larvae were perceived, probably because of the greater size of the larvae. Tentorial pits were usually more pronounced, and rows of spinules over food basket area were visible. Additionally, the maxillary palpus of male larvae were slightly longer and paxilliform-shaped, due to the presence of a well-develop enclosed apical sensillum. Similarly, the galea acquired a doubled-elevation because of the enlarged apical sensilla. The first thoracic spiracle of last instar male larvae is much larger than the remaining ones. Gyne Larvae (Figure 5A-D): Antennal sensillae more pronounced and setaceous. Tentorial pits clearly discernible on cranium. Hypopharynx more densely spinulose. Food basket area with rows of short spines. Pupae (Figure 7A-C): Young pupae are yellowish white, getting darker as they mature into imagoes. Always exarate and without cocoons. Worker pupae (Figure 7A) varied between 2-4 mm long, while male pupae (Figure 7B) averaged 4.2 mm, and gyne pupae (Figure 7C) were usually around 5.5 mm long. Intraspecific variations: From analyzing numerous last instar larvae of S. saevissima and S. invicta we were able to observe frequent intraspecific variation in the morphology of head setae (i.e. ‘hairs’ according with the terminology of the Wheelers), in which occipital and vertex hairs can alternate between simple and bifid, and often even 3-branched morphology at apparently random positions. Variations occurred among specimens of the same geographical region and colony. It is worth noting that some specimens of S. saevissima and S. invicta had head hairs all simple. Discussion This is the first detailed description of the immature stages of S. saevissima, and the first ant larval description to include specimens of all castes and from different geographical locations. Younger first- and second-instar larvae were always found in low frequencies in the collected nests, suggesting that they might last only a few hours. Other 53 possible explanations for they being difficult to obtain would be that they are placed in some particular region of the nest which is more difficult to collect by the usual methods or that they are not efficiently recovered by flotation. This can only be answered by direct experimentation and observation of the duration of each larval instar. The fact the reproductive larvae are basically identical to worker larvae confirms previous impressions stated by WHEELER; WHEELER (1976). These authors mentioned being only able to sort reproductive from worker ant larvae when they were at the last instar, as reproductive larvae acquired considerably greater bodily proportions. The distinct body shape acquired by the sexual larvae of males and gynes of S. saevissima made sexual separation quite easy. This alteration of shape is certainly caused by the developing pupa inside. The enlarged thoracic spiracle of male prepupae probably relates with intense metabolism in that area, e.g. development of powerful flight muscles. This and the apparently thicker integument of male larvae merit direct investigation by serial dissections of whole larvae. The larval instars of S. invicta were previously described by ONEIL & MARKIN (1975), who also presented descriptions of larvae of all castes. The larval instars were later revisited by PETRALIA; VINSON (1979) who added SEM images of all stages and presented detailed descriptions appointing flaws in the original description by ONEIL; MARKIN (1975), as for instance in the morphology of the younger larvae which were originally described to have bifid hairs (see ONEIL; MARKIN, 1975). We find it very unlikely that such striking differences would have been due to intraspecifc variation, as they were never reported again by other authors. Although ONEIL; MARKIN (1975) claimed that larvae of different castes had head capsules of significantly different width, our present observations with S. saevissima and brief observations with the morphologically identical S. invicta do not support this assertion. We cannot but wonder about the origin of the unconfirmed observations of ONEIL; MARKIN (1975), but our images and those of PETRALIA; VINSON (1979) leave little room for speculation. In a recent revision of morphological characters and phylogenetic relationships within fire ants, PITTS et al. (2005) proposed the use of the morphology and configuration head setae of fourth instar larvae to support species separation. The head hairs to be used are those above the antennal level, individualized as “first and second row on vertex” and “occipital row”. According with PITTS et al. (2005), all 54 these head setae should be bifid among fire ant larvae. However, we found considerable variation in this pattern within S. saevissima, with many instances of specimens with the medial head hairs above antennal level being without ramifications (simple). By relying on the larval characters proposed by PITTS et al. (2005), one would have taken the specimen of Fig. 1 for Solenopsis megergates, and most of the other specimens for S. invicta. Similar variation in the morphology of head hairs was also recently observed in Paratrechina longicornis Latreille (FOX et al., 2007). The observed intraspecific variation of head hair morphology was not reported by previous authors (WHEELER; WHEELER, 1951; PITTS et al., 2005), probably because of their limited sample size. The find is solid evidence that head hair morphology is not reliable as a character for sorting between fire ant species. From comparing the last instar larvae of typical S. saevissima with those of S. invicta, we were not able to find any differences that would be useful to distinguish between these two species. It seems to us that mature larvae are not useful for species separation in fire ants, but investigations with numerous larvae of further fire ant species are needed to confirm this. Maybe there are some shared patterns among some species that would aid species separation, but truly specific patterns already seem unlikely. Finally, the present description adds to the body of knowledge of ant immature stages, while presenting SEM images of all castes for the first time. Some of the observed traits found may have taxonomical importance, and probably reflect specializations to the life habits of the group. We do not recommend the use of fire ant larvae for species separation as the morphological characters proposed exhibited considerable intraspecific variation in two of the most common fire ant species. Cited References BOLTON, B.; ALPERT, G.; WARD, P. S.; NASKRECKI, P. Bolton catalogue of the Ants of the World: 1758-2005. Harvard University Press, Cambridge, CD-ROM. 2006. FINLAYSON, T. A classification of the subfamily Pimplinae (Hymenoptera: Ichneumonidae) based on final-instar larval characteristics. Canadian Entomologist, v. 99, p. 1-8, 1975. 55 FOX, E. G. P.; SOLIS, D. R.; JESUS, C. M.; BUENO, O. C.; YABUKI, A. T.; ROSSI, M. L. On the immature stages of the crazy ant Paratrechina longicornis (Latreille 1802) (Hymenoptera: Formicidae). Zootaxa, v. 1503, p. 1-11, 2007. O'NEAL, J.; MARKIN, G. P. The larval instars of the imported fire ant Solenopsis invicta (Hymenoptera: Formicidae). Journal of Kansas Entomological Society, v. 48, pp. 141–151, 1975. PETRALIA, R. S.; VINSON, S. B. Developmental morphology of larvae and eggs of the imported fire ant, Solenopsis invicta. Annals of the Entomological Society of America, v. 72, p. 472–484, 1979. PITTS, J. P.; HUGH, M. C. J.; ROSS, K. G. Cladistic analysis of the fire ants of the Solenopsis saevissima species-group. Zoologica Scripta, v. 34, p. 493 –505, 2005. SCHULTZ, T. R.; MEIER, R. A phylogenetic analysis of the fungus-growing ants (Hymenoptera: Formicidae: Attini) based on morphological characters of the larvae. Systematic Entomology, v. 20, p. 337-370, 1995. TRAGER, J. C. A revision of the fire ants of the Solenopsis geminata Group (Hymenoptera: Formicidae: Myrmicinae). Journal of the New York Entomological Society, v. 99, p. 141-198, 1991. WHEELER, G. C.; WHEELER, J. The ant larvae of the myrmicine tribe Solenopsidini. American Midleast Naturalist, v. 54, p. 119-141, 1951. WHEELER, G. C.; WHEELER, J. The ant larvae of the myrmicine tribe Leptothoracini. Annals of the Entomological Society of America, v. 48, p. 17-29, 1955. WHEELER G. C.; WHEELER, J. Ant larvae: review and synthesis. Memories of the Entomological Society of Washington, v. 7, p. 1-108, 1976 56 Figure 1. Egg and first instar larva of Solenopsis saevissima. A. Egg. B. Hatching larva. C. Side view of first instar larva; inlet = thoracic spiracle. D. Head capsule and mouthparts; lb = labrum; md = mandible; mx = maxilla; lm = labium. E. mandible. F. Maxilla. G. Labium. 57 Figure 2. Second instar larva of Solenopsis saevissima. A. Larva on side view; inlet = thoracic spiracle. B. Head capsule. C. Mouthparts; arrows = spines around mouth entrance. D. Larva moulting to third instar. 58 Figure 3. Third instar larva of Solenopsis saevissima. A. Full larva in frontal view. B. Mouthparts; md = mandible; mx = maxilla; lb = labium; arrow = spiny papillae at mouth entrance; middle inlet = thoracic spiracle. C. Larva moulting to fourth instar. 59 Figure 4. Fourth instar larva of Solenopsis saevissima. A. Full body in side view; central inlet = thoracic spiracle. B. Head capule in frontal view; upper inlet = antennal sensilla. C. Lower mouthparts; arrow = spines on lower portion of labrum; mx = maxilla; Lb = labium. D. Frontal view of left mouthparts; md = mandibule; Lr = labrum; mx = maxilla. 60 Figure 5. Last instar larva of gyne of Solenopsis saevissima. A. Full body in frontal view; lower inlet = thoracic spiracle. B. Head capsule in frontal view, upper inlet = antennal sensilla. C. Mouthparts in side view; Lr = labrum; md = mandibule; mx = maxilla. D. Food basket area, just under lower mouthparts (indicated with *); arrows = rows of spines on integument. 61 Figure 6. Last instar larva of male of Solenopsis saevissima. A. Full body in side view. B. Head capsule in frontal view. C. Mouthparts in frontal view; Lr = labrum; mx = maxilla; mb = mandible. D. Closer view on right maxilla, showing maxillary palp and galea. E. Closer view on frons of head capsule; arrows = tentorial pits on upper limits of the clypeus. 62 Figura 7. Pupae of major worker (A), male (B), and gyne (C) of Solenopsis saevissima. 63 Figure 8. Intraspecific variation in the morphology of head hairs of fourth instar larvae of Solenopsis saevissima. All head hairs above antennal level – indicated by a white line – in larvae of this species ought to be bifid. However, the superior central hairs on the head displayed to the left are all bifid, whist simple on the head to the right. CAPÍTULO 4 MORFOLOGIA GERAL E ULTRAESTRUTURA DO APARATO DE VENENO E GLÂNDULA CONVOLUTA DA FORMIGA LAVA-PÉS Solenopsis saevissima Smith Journal of Insect Science, n. 24, 2010 � 65 General morphology and ultrastructure of the venom apparatus and convoluted gland of the fire ant, Solenopsis saevissima Smith Abstract A group of 13 species of the genus Solenopsis is markedly difficult to assess taxonomically, although they are of considerable economical and medical importance in some countries where some of them were introduced. These ants are aggressive and their venomous stings can be very allergenic. The venom apparatus has been described in fine detail for only two of these species, and differences in this structure among the different species might prove useful as taxonomic characters. The venom apparatus of Solenopsis saevissima Smith (Hymenoptera: Formicidae) is herein described with the aid of light and electron microscopy techniques, and compared to that of S. invicta and S. richteri. The cellular organization of the different parts present differences that suggest functional specialization. In general, the different tissues were abundant in vesiculae and mitochondria, but presented little endoplasmic reticulum and few ribosomes, probably because they produce little protein. The length of the free filaments of the venom gland and the width of their internal ducts seems to vary from what was described for S. richteri, but this may be of little use to taxonomy. � 66 Introduction Venom apparatuses are common structures of hymenopterans and are involved in the production of active compounds to be delivered through an ovipositor or sting. Many hymenopterans have stings, which, apart from being used to subdue their prey, can be used effectively for defense. In some ants, the sting is used for colony defense, and some people can develop serious anaphylactic reactions to ant venoms (BROWN & HEDDLE 2003). Some ants of the genus Solenopsis Westwood (Hymenoptera: Formicidae) are known as fire ants (VINSON, 1986) because of their painful stings. They aggressively attack in swarms when their fragile, earthen nests are disturbed. Fire ants are native to the Americas and most diverse in South America, but some species of this group have been shipped and introduced into other world regions inadvertently. At least one species, Solenopsis invicta Buren, has become a major public concern, mainly in the United States, because of its marked adaptability to human environments and the allergenicity of its sting (RHOADES et al., 1989; DESHAZO; BANKS, 1994; DESHAZO; WILLIAMS, 1995; deShazo et al., 1999; Kemp et al., 2000). One species, Solenopsis saevissima Smith, is still restricted to South America and common in Brazil (ROSSI & FOWLER 2004). It has not been studied as extensively as S. invicta. Both species belong to a particularly problematic ant group, in terms of taxonomy and systematic, known as the “Solenopsis saevissima group of species” (PITTS et al., 2005). It includes 13 fire ant species that exhibit marked morphological similarity and intraspecific variability. Some species are capable of hybridization, rendering most morphological characters for species separation unreliable (VANDER MEER, 1985; PITTS et al., 2005). There is still some ongoing discussion about the validity of these species and the best characters to be used in defining each species (ROSS & TRAGER, 1990; ROSS; SHOEMAKER, 2005). The venom apparatus of Solenopsis richteri Forel was thoroughly described, including histological aspects, by CALLAHAN et al. (1959). Later, the venom apparatus of S. invicta, a similar species with which S. richteri can hybridize (VANDER MEER, 1985), was briefly described by BILLEN (1990), who also analyzed some ultrastructural aspects of it. No other venom apparatuses of any species of this group have been described, but it is well known that the venoms of the different species of fire ants have distinct chemical composition (JONES; BLUM, 1982; FOX; PALMA; BUENO, � 67 unpublished results). The different compositions might reflect differences in the internal organization of the structures of the ven