FORENSE, Chrysomya Megacephala (F.) E Chrysomya Putoria (W.) (DIPTERA: CALLIPHORIDAE), SOB INFLUÊNCIA DE DIFERENTES TEMPERATURAS E / OU CLORIDRATO DE FLUOXETINA

MARCELA AQUIYAMA ALONSO

DESENVOLVIMENTO DE IMATUROS DE ESPÉCIES DE IMPORTÂNCIA FORENSE, Chrysomya megacephala (F.) E Chrysomya putoria (W.) (DIPTERA: CALLIPHORIDAE), SOB INFLUÊNCIA DE DIFERENTES TEMPERATURAS E / OU CLORIDRATO DE FLUOXETINA

CAMPINAS 2015

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RESUMO

Os insetos constituem o grupo mais diversificado e abundante do Reino Animal, com ampla diversidade morfológica, fisiológica e de hábitos alimentares e por isso podem ser encontrados em vários habitats e ecossistemas. Insetos necrófagos podem ser vestígios e fontes de informações de interesse forense, como para a estimativa do intervalo pós-morte (IPM), baseada, por exemplo, na idade dos imaturos que se criam em corpos em decomposição. O desenvolvimento desses insetos pode ser afetado pela variação de temperatura e presença de substâncias tóxicas nos tecidos de um cadáver, entre outros fatores. Chrysomya megacephala (F.) e Chrysomya putoria (W.) (Diptera: Calliphoridae), introduzidas no Brasil, são consideradas de importância forense, médico e veterinária, devido aos seus comportamentos sinantrópicos e necrófagos. No presente estudo foi avaliado o tempo de desenvolvimento de imaturos na fase embrionária e pós-embrionária de C. megacephala e C. putoria sob diferentes temperaturas e / ou presença de cloridrato de fluoxetina, um antidepressivo, em fígado de coelho, o substrato alimentar. A relação entre temperatura e desenvolvimento, na fase embrionária, foi similar entre ambas as espécies. O tempo de desenvolvimento dos ovos para C. megacephala variou aproximadamente de 64-7h a 13 e 35 °C, respectivamente, e para C. putoria de 69-8h a 13 e 35 °C, respectivamente. Houve eclosão de larvas a 13 °C, mas as mesmas não completaram o desenvolvimento. A temperatura e o cloreto de fluoxetina afetaram o desenvolvimento dos imaturos, na fase na pós-embrionária. Para ambas as espécies, as larvas do grupo controle completaram seu desenvolvimento 24h mais rápido que o grupo com fluoxetina a 17 °C, mas apresentaram o desenvolvimento 12h mais lento a 35 °C. Estudos considerando tempo real de desenvolvimento dos ovos e avaliando como a combinação de duas ou mais variáveis podem influenciar o desenvolvimento de insetos de interesse forense são de grande valia para aumentar a acurácia da estimativa do IPM.

Palavras-chave: Entomologia forense, Entomotoxicologia, Intervalos pós-morte, Insetos necrófagos

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ABSTRACT

Insects are the most diverse and abundant group of the Animal Kingdom, with great diversity of morphological, physiological and feeding habits and are found in nearly all habitats and ecosystems. Scavengers species can provide important information of forensic interest, as the postmortem interval (PMI) estimate, based on, e.g., the age of the larvae reared in decomposing bodies. The development of these insects can be affected by temperature oscillation and presence of toxic substances, among other factors, on the rearing media. Chrysomya megacephala (F.) and Chrysomya putoria (W.) (Diptera: Calliphoridae), introduced in Brazil, are considered of forensic, medical and veterinary importance, due to their necrophagous and synanthropic behaviour. This study evaluated the developmental time of C. megacephala and C. putoria under different temperatures and / or with fluoxetine hydrochloride, an antidepressant drug, in rabbit liver, the rearing substrate. The relationship between temperature and development, on the embryonic phase, was similar for both species. Egg developmental time for C. megacephala was approximately of 64-7h at 13 and 35 °C, respectively, and for C. putoria was 69-8h at 13 and 35 °C, respectively. The larval hatching occurred at 13 °C, but, at this temperature, the larval development was not completed. Both temperature and fluoxetine hydrochloride, when present, affected the development of the larvae. For both species, the larvae of control group completed their development 24h faster than the fluoxetine hydrochloride group at 17 °C, but the development was 12h slower at 35 °C. Studies considering real egg developmental time and evaluating, simultaneously, the insects’ response for two or more variables that might influence their development are of great value to increase the accuracy of PMI estimate.

Key-words: Forensic entomology, Entomotoxicology, Post-mortem interval, Necrophagous insects

SUMÁRIO AGRADECIMENTOS ...... XIII LISTA DE FIGURAS ...... XV LISTA DE TABELAS ...... XVII 1. INTRODUÇÃO ...... 1 2. REVISÃO BIBLIOGRÁFICA ...... 2 2.1. CALLIPHORIDAE ...... 2

2.2. ENTOMOLOGIA FORENSE ...... 2

2.3. INTERVALO PÓS-MORTE E GRAU-HORA ACUMULADO ...... 4

2.4. ENTOMOTOXICOLOGIA ...... 6

2.5. CLORIDRATO DE FLUOXETINA ...... 6

3. OBJETIVOS...... 8 4. CAPÍTULO I – EGG DEVELOPMENTAL TIME AND SURVIVAL OF Chrysomya megacephala (F.) AND Chrysomya putoria (W.) (DIPTERA: CALLIPHORIDAE) UNDER DIFFERENT TEMPERATURES ...... 9 4.1. ABSTRACT ...... Erro! Indicador não definido.

4.2. RESUMO ...... Erro! Indicador não definido.

4.3. INTRODUCTION ...... 11

4.4. MATERIALS AND METHODS ...... 12

4.5. RESULTS ...... 14

4.6. DISCUSSION ...... 14

4.7. ACKNOWLEDGEMENTS ...... 16

4.8. REFERENCES CITED ...... 17

5. CAPÍTULO II – EFFECT OF DIFFERENT TEMPERATURES AND PRESENCE OF FLUOXETINE HYDROCHLORIDE ON THE DEVELOPMENT OF FORENSIC IMPORTANCE SPECIES Chrysomya megacephala (F.) AND Chrysomya putoria (W.) (DIPTERA: CALLIPHORIDAE) ...... 26 5.1. ABSTRACT ...... 26

5.2. RESUMO ...... 27

5.3. INTRODUCTION ...... 28

5.4. MATERIALS AND METHODS ...... 29

5.5. RESULTS AND DISCUSSION ...... 31

5.6. CONCLUSIONS ...... 43

5.7. ACKNOWLEDGEMENTS ...... 43

5.8. REFERENCES ...... 43

6. CONCLUSÕES GERAIS ...... 46 7. REFERÊNCIAS BIBLIOGRÁFICAS ...... 47 8. ANEXO ...... 55

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AGRADECIMENTOS

Agradeço aos meus pais Maria Lúcia e Paulo pelo carinho, pela compreensão e por apoiarem todas as escolhas que fiz. Aos meus irmãos Bruna e André por sempre me acolherem em São Paulo com as refeições mais gostosas e divertidas. E à minha família pelo apoio em todos os aspectos possíveis. Agradeço aos amigos de Rio Preto pelos momentos inesquecíveis, divertidos e tantos outros carnavais, essenciais para manter a energia necessária nessa jornada. Em especial àqueles que além de legais são lindos de corpo e alma, sempre com um colo disponível nos momentos complicados e longe da família. Agradeço aos amigos da RepLatrô e agregados, minha família em Campinas, pelo carinho, paciência e alegria nos momentos bons e ruins, desde a época da faculdade. Pelas noites em claro estudando na sala, pelos “almoços em família” e estradas percorridas. Também ao Bill, minha alegria de chegar em casa todos os dias. Agradeço meus amigos da Unicamp pela singularidade de cada um, essenciais para minha formação, tanto pessoal quanto profissional. Agradeço àqueles amigos especiais da Bio08D por me acompanharem de perto numa fase de tantas mudanças e descobertas. Obrigada pelo amor e cumplicidade, pra sempre. E também aos amigos do Conds Rambo, pela saúde física e mental. Agradeço aos amigos e companheiros de trabalho do L2B pelo conhecimento adquirido, pelos litros de café e muitos docinhos, pela ajuda nos experimentos e por tornarem nosso ambiente tão “gostouso”, divertido e musical! Também aos professores e técnicos do departamento de Biologia Animal – Parasitologia pela disponibilidade para ajudar e contribuir com conhecimento sempre que necessário. Agradeço muito aos técnicos do Núcleo de Medicina Experimental pela tranquilidade e leveza que trouxeram para a parte experimental que seria de maior desafio emocional pra mim. Agradeço aos professores que participaram da qualificação e pré-banca, por contribuírem com o meu trabalho. Ao Prof. Arício por toda a ajuda e disponibilidade, por me receber no laboratório e mostrar o mundo da Entomologia. Ao Prof. Cláudio Von Zuben participar da banca. Agradeço à Profa. Patricia pela orientação, por responder todas minhas (muitas, muitas e muitas) perguntas e me fazer acreditar que ia dar certo. Agradeço também ao programa de pós-graduação em Biologia Animal e à Fapesp pelo apoio financeiro.

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LISTA DE FIGURAS

CAPÍTULO I

Figure 1. Egg survival for C. megacephala (F.) and C. putoria (W.) at eight temperatures. The equations that represents the survival are, for C. megacephala: y = -0.4021 + 0.0590x - 0.0006x2; R2 = 0.75, and for C. putoria: y = -0.6293 + 0.1002x - 0.018x2; R2 = 0.68. The P-values are based on the Mann-Whitney test for comparisons of the egg survivor of the two species in each tested temperature...... 23 Figure 2. Temperature (T) and Duration of development (D) of C. megacephala (F) and C. putoria (W.). The regression lines are used to determine t and K for egg development for each species. 24 Figure 3. Developmental time at different temperatures for C. megacephala (F.) and C. putoria (W.) data here presented and published data. 1- Greenberg and Szyska 1984; 2- Gabre et al. 2005; 3- Prins et al. 1982; 4- Barros-Cordeiro and Pujol-Luz 2010; 5- Wells a and Kurahashi 1994. ... 25

CAPÍTULO II

Figure 4. Example of Chrysomya megacephala (F.) (Diptera: Calliphoridae) body length measurement with stereomicroscope and image capture system...... 30 Figure 5. Chrysomya megacephala (F.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were A = 13 ±1 °C and B = 17 ±1 °C. All larvae died at 13 °C before reach minimum weight (0.002 g), therefore there is no SD for the temperature. Data analysis with an overall error rate (α) of 0.05...... 34 Figure 6. Chrysomya megacephala (F.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were C = 20 ± 1 °C and D = 25 ±1 °C. Data analysis with an overall error rate (α) of 0.05...... 35 Figure 7. Chrysomya megacephala (F.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were E = 30 ± 1 °C and F = 35 ±1 °C. Data analysis with an overall error rate (α) of 0.05...... 36

Figure 8. Chrysomya putoria (W.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were G= 13 ±1 °C and H = 17 ±1 °C. All larvae died at 13 °C before reach minimum weight (0.002 g) therefore there is no SD for the temperature. Data analysis with an overall error rate (α) of 0.05...... 37 Figure 9. Chrysomya putoria (W.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were I = 30 ±1 °C and J = 35 ±1 °C. Data analysis with an overall error rate (α) of 0.05...... 38 Figure 10. Chrysomya putoria (W.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were L = 30 ±1 °C and M = 35 ±1 °C. Data analysis with an overall error rate (α) of 0.05...... 39

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LISTA DE TABELAS

CAPÍTULO I

Table 1. Mean number of eggs ± standard deviation (SD), incubation time (hour) and egg survival (%) of Chrysomya megacephala (F.) and Chrysomya putoria (W.) (Diptera: Calliphoridae) at eight temperatures...... 22

CAPÍTULO II

Table 2. Duncan multiple comparisons test for Chrysomya megacephala (F.) and Chrysomya putoria (W.) (Diptera: Calliphoridae) development at different temperatures with weight and body length as response variables. The means with the same letter are not different. The small letters are of comparisons inside the column, between the different temperatures inside the control or the fluoxetine group. The capital letters are of comparisons in the lines, between the weight or body length from the control and the fluoxetine group inside the same temperature. Bold letters indicates the means with statistical differences. Data analysis with an overall error rate (α) of 0.05...... 40

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1. INTRODUÇÃO

Os insetos (Hexapoda, Insecta), representam cerca de 60% das espécies descritas e constituem o grupo mais diversificado e abundante do Reino Animal, com aproximadamente um milhão de espécies descritas (Rafael et al. 2012, Zhang 2013). Podem estar presentes tanto nos habitats terrestres quanto aquáticos, ecossistemas naturais e antrópicos e possuem ampla diversidade morfológica, fisiológica, de ciclos biológicos e de hábitos alimentares (Chapman 1998, Gullan e Cranston 2007, Rafael et al. 2012). Muitos Hexapoda estão associados ao homem de modo harmônico ou causando algum tipo de prejuízo. Podem estar associados à agricultura e alimentação como polinizadores, pragas, produtores de matérias-primas, tais como mel e seda, no controle biológico de pragas ou mesmo como fonte de alimento. No âmbito médico-veterinário os insetos podem ser vetores ou agentes de doenças; fonte de matéria-prima para indústria de medicamentos e cosméticos (Triplehorn e Johnson 2004, Gullan e Cranston 2007), além de serem usados diretamente tratamento de doenças, como na terapia larval, método que usa imaturos de moscas para limpeza de feridas de pele de difícil cicatrização (Sherman 2000). Os insetos também podem ser fonte de informações para elucidação de casos da área forense (Keh 1985, Amendt et al. 2004). A partir de seus hábitos e ciclo de vida é possível determinar, por exemplo, quando e/ou onde ocorreu a infestação por insetos, ou partes deles, e o responsável pela falta de integridade do alimento ou bem material em questão (Lord e Stevenson 1986). Também é possível determinar o tempo decorrido entre a morte de uma pessoa até o momento que o corpo foi encontrado (Greenberg e Kunich 2002, Byrd e Castner 2010). A idade de imaturos de moscas que se desenvolvem em um corpo em decomposição é um dos parâmetros usados para determinar o tempo de infestação e, consequentemente, da morte investigada (Smith 1986, Hall 1990). No entanto, o desenvolvimento das larvas pode ser influenciado por fatores como: temperatura, umidade, órgão que foi usado como alimento e substâncias tóxicas ingeridas pelo indivíduo antes da morte (Goff e Lord 1994, Chapman 1998, Niederegger et al. 2013). Assim, o conhecimento prévio da resposta dos imaturos a esses fatores é crucial para garantir a acurácia desejada na estimativa do tempo decorrido após a morte.

2. REVISÃO BIBLIOGRÁFICA

2.1. CALLIPHORIDAE

A ordem Diptera é a quarta maior dentro da Classe dos insetos, com cerca de 160.590 espécies descritas (Zhang 2013), e as famílias mais comumente encontradas e que possuem maior aplicação como evidência na área forense são: Calliphoridae, Sarcophagidae e Muscidae (Byrd e Castner 2010). Os Calliphoridae possuem importância na reciclagem da matéria orgânica em decomposição, são vetores de patógenos, podem causar miíases ou serem usados na terapia larval, além de estarem entre os primeiros animais a entrar em contato com um corpo em decomposição (Zumpt 1965, Guimarães e Papavero 1999, Carvalho et al. 2000). Tais corpos são importante fonte de proteína para o desenvolvimento dos imaturos destes insetos, principalmente, e para desenvolvimento dos folículos ovarianos dos adultos (Nuorteva, 1977, Smith 1986). As fêmeas, em sua maioria, colocam 300 ovos por postura, podendo produzir cerca de 3000 ovos ao longo da vida (Amendt et al. 2004). O gênero Chrysomya (Robineau-Desvoidy 1830) (Diptera: Calliphoridae) caracterizado por adultos de aspecto metálico, é de interesse forense pela abundância com que é encontrado, tanto na forma adulta como quanto imatura, alimentando-se em cadáveres (Carvalho et al. 2000). Chrysomya megacephala (Fabricius 1794) e Chrysomya putoria (Wiedemann 1830) (Diptera: Calliphoridae) são duas espécies acidentalmente introduzidas no Brasil (Guimarães et al. 1978) e que foram encontradas se criando em corpos na Paraíba, Pernambuco, Rio de Janeiro e São Paulo (Carvalho et al. 2000, Oliveira-Costa et al. 2001, Andrade et al. 2005, Oliveira e Vasconcelos 2010). Pelo interesse médico, veterinário e forense, a biologia e distribuição dessas espécies é amplamente estudada, tanto no Brasil (Barros-Cordeiro e Pujol-Luz 2010) quanto em outros países como: África do Sul (Richards e Villet 2009, Richards et al. 2009) Egito (Gabre et al. 2005) e Estados Unidos (Wells 1991).

2.2. ENTOMOLOGIA FORENSE

A entomologia forense consiste no estudo dos insetos e outros artrópodes associados a procedimentos periciais com propósito principal de levantar informações e vestígios que possam

2 auxiliar um processo investigativo (Amendt et al. 2004, Goff 2010, Thyssen 2011). Lord e Stevenson (1986) a dividiram em três categorias: - urbana: referente a danos estruturais em construções ou eletro-eletrônicos particulares ou patrimônio público causados por insetos, infestações de cupins, baratas e outros insetos causadores de problemas para humanos. Apesar do nome não está restrita ao ambiente urbano; - de produtos estocados: focado principalmente no controle de insetos que se alimentam de grãos e causam prejuízo para a indústria alimentícia, e na infestação de alimentos processados por insetos ou partes deles, a fim de determinar o responsável, na cadeia de produção, comercialização e consumo do produto, pela má conservação do mesmo; - médico-criminal: com enfoque nos danos a animais, de criação ou pet, na transmissão de doenças, miíases e nos casos de negligência a idosos, crianças e pessoas que necessitam de cuidados especiais, além de auxiliarem nas respostas a quesitos da perícia criminal. No âmbito médico-criminal, estudos buscam fornecer dados biológicos e desenvolver técnicas para colaborar com o esclarecimento de questões sobre: o local de óbito e se houve deslocamento do corpo, usando a ecologia, distribuição geográfica e endemismo das espécies; o modo da morte, no caso de suspeita de abuso de substâncias tóxicas, de envenenamento ou indicando presença de feridas ou pólvora e, ainda, o tempo aproximado entre o início da colonização do corpo por insetos e o momento que o corpo foi encontrado, estimando-se assim a quanto tempo ocorreu a morte, considerando o desenvolvimento dos insetos necrófagos e a sucessão de espécies encontradas no corpo (Nuorteva 1977, Smith 1986, Goff et al. 1989, Hall 1990, Catts e Goff 1992, Introna et al. 2001, Byrd e Castner 2010). Casos de negligência, com animais, crianças, idosos e outras pessoas que necessitam de cuidados de terceiros, também podem ser esclarecidos com base nos hábitos alimentares e no tempo de desenvolvimento dos insetos que infestam o organismo e seu ambiente (Zumpt 1965). Insetos podem ser usados como amostra para a detecção de substâncias tóxicas no organismo usado como substrato alimentar. A entomotoxicologia é a área da entomologia forense que estuda os métodos usados na detecção de substâncias em insetos e como estas podem afetam seu desenvolvimento (Nuorteva e Nuorteva 1982, Introna et al. 2001).

2.3. INTERVALO PÓS-MORTE E GRAU-HORA ACUMULADO

O IPM consiste no tempo decorrido entre a morte até o momento em que o corpo é encontrado e pode ser estimado a partir do padrão de mudanças físico-químicas que ocorrem em um corpo após a morte, tais como: esfriamento, perda de massa, rigidez cadavérica, livores hipostáticos, crioscopia do sangue, reação muscular, alteração no nível de potássio no humor vítreo, entre outros (França 2004). O processo de decomposição também pode ser caracterizado por fases consecutivas, cada uma apresentando sua cronologia própria, tentando atender aos fins de estimar o IPM: coloração, gasosa, coliquativa e esqueletização (Reed 1958, Jirón e Cartín 1981). Outra forma de estimar o IPM é através da sucessão de espécies de artrópodes que colonizam um corpo (Mégnin 1894), ou por meio da idade dos imaturos de insetos que ali se alimentam (Catts e Goff 1992), sendo em ambos os casos o IPM definido como o intervalo decorrido entre o início da colonização do corpo até sua descoberta. A estimativa do IPM feita a partir da sucessão ecológica é baseada no princípio de que a atratividade de um corpo em decomposição, para os insetos necrófagos, varia com o tempo em decorrência das mudanças químicas inerentes ao processo e, assim, pressupõe-se que a colonização por diferentes espécies deva ocorrer dentro de uma sequência ou ordem previsível (Amendt et al. 2004). O conjunto de espécies encontradas no corpo fornece o tempo máximo de exposição do corpo, sendo assim classificado como IPM máximo (IPMmáx), mas essa estimativa está sujeita à dificuldade de avaliar o comportamento dos insetos que são encontrados neste tipo de recurso. Schoenly (1992) demonstrou que insetos necrófagos apresentam dois comportamentos de sucessão: os que persistem na carcaça por um período e aqueles que aparecem, abandonam e voltam a aparecer na carcaça, o que dificulta a previsão das ondas de sucessão com a precisão adequada. A idade dos imaturos de insetos que se criam nos corpos, outro parâmetro usado na estimativa do IPM, é baseada no comprimento ou na massa corpórea dos indivíduos (Greenberg e Kunich

2002) e é usada para estimar o IPM mínimo (IPMmin). Tal estimativa equivale ao tempo mínimo que o corpo ficou exposto a condições propícias para a colonização por insetos. E, geralmente, os Diptera encontro o corpo e depositam ovos apenas alguns minutos após a (Catts 1992, Campobasso et al. 2001). O cálculo do IPMmin é feito através da fórmula da constante térmica (K), expressa em graus-horas ou graus-dias acumulados, que pode ser calculado das seguintes maneiras: K = tempo de desenvolvimento × (temperatura de desenvolvimento – limar térmico inferior) ou tempo de

4 desenvolvimento × temperatura = K + limiar térmico inferior × tempo de desenvolvimento, que levam em conta as necessidades térmicas de cada espécie, bem como a temperatura na qual os insetos se desenvolveram e é baseada na dependência da temperatura para taxa de desenvolvimento dos animais poiquilotérmicos (Wigglesworth 1972, Wagner et al. 1984, Haddad et al. 1999, Ikemoto e Takai 2000, Greenberg e Kunich 2002). Ainda, cada evento do desenvolvimento de um inseto, como eclosão das larvas, mudança de instar ou pupariação pode possuir um número de graus-hora acumulado associado (Byrd e Castner 2010). Assim, para esta estimativa é preciso ter acesso aos valores teóricos de K e limiar térmico inferior da espécie e fase de desenvolvimento do espécime utilizado que são obtidos através de dados empíricos e usando-se modelos de regressão lineares ou não lineares. O modelo da K foi proposto por muitos autores e compilado em Wagner e colaboradores (1984).

Tanto para o cálculo do IPMmáx quanto do IPMmin, um entomologista forense precisa de um bom conhecimento em taxonomia, para correta identificação das espécies em diversas fases de desenvolvimento. Também é necessário ter acesso a dados sobre a biologia e comportamento de insetos necrófagos, que podem ser influenciados tanto por fatores abióticos como temperatura de exposição, umidade relativa, fotoperíodo e latitude (Hanski 1977, Wells e Kurahashi 1994, Mello et al. 2012, Nassu et al. 2014), quanto pela presença de substâncias tóxicas nos tecidos do cadáver, pelas caraterísticas nutricionais do meio de desenvolvimento, pela densidade larval e competição inter e intraespecífica (Ullyett 1950, Hanski 1977, Goff et al. 1989, Goodbrood e Goff 1990, Wells e Greenberg 1992, Goff e Lord 1994, Reis et al. 1996, Von Zuben et al. 2000, Souza et al. 2011, Niederegger et al. 2013, Rezende et al. 2014). Medidas da temperatura de desenvolvimento dos imaturos são indispensáveis para o cálculo do IPM, e são geralmente obtidas de estações meteorológicas próximas ao local onde o corpo foi encontrado (Johnson et al. 2012) e não do local em si. Entretanto, os imaturos que se alimentam de tecidos em decomposição apresentam comportamento gregário, da eclosão à fase de pré-pupa, e devido ao número de ovos depositados por fêmea e à oviposição agregada, é comum observar massas de centenas de imaturos nos corpos colonizados (Turner e Howard, 1992, Slone e Gruner 2007). O habito gregário, juntamente com a movimentação o metabolismo dos imaturos, comumente leva a um aumento de temperatura, chamado efeito de massa larval (Campobasso et al. 2001, Charabidze et al. 2011). Esse microclima gerado pela massa larval protege os imaturos de possíveis quedas bruscas de temperatura (Campobasso et al. 2001) e pode apresentar

temperatura até 20 °C acima da temperatura ambiente (Turner e Howard 1992). Métodos não invasivos para medidas de temperaturas, como imagem infravermelha, aumentam a acurácia da estimativa de temperatura do microclima de desenvolvimento das larvas, e consequentemente da estimativa do IPM, e auxiliam estudos sobre os fatores que contribuem para a geração de calor na massa larval e conhecimento da biologia da espécie (Johnson e Wallman 2014).

2.4. ENTOMOTOXICOLOGIA

A entomotoxicologia estuda a uso de insetos necrófagos como amostras alternativas na detecção de substâncias tóxicas presentes no substrato alimentar, principalmente quando não há disponibilidade de tecidos corporais para análise. Esse campo da entomologia também avalia se a presença de substâncias tóxicas nos tecidos de um cadáver pode alterar o comportamento e a taxa de desenvolvimento das larvas que dele se alimentam e a atração dos insetos necrófagos (Goff e Lord 1994, Bourel et al. 1999, Hédouin et al. 1999, Introna et al. 2001, Gosselin et al. 2011). Estudos com fenobarbital, benzodiazepínicos, anfetaminas, escopolamina, esteroides anabólico- androgênicos, cocaína, quetamina e metadona demonstraram influência significativa ou não no desenvolvimento de imaturos de espécies de interesse forense e podem servir de referência para investigações no âmbito médico-legal (Carvalho et al. 2001, Ferrari et al. 2008, Grella et al. 2007, Lü et al. 2014, Mullany et al. 2014, Oliveira et al. 2009, Souza et al. 2011, Rezende et al 2014). Insetos necrófagos são encontrados em grande quantidade e distribuídos por diferentes órgãos e partes do corpo, então o fígado, músculo e a região da cabeça são recomendados para coleta de espécimes usados na detecção de substâncias tóxicas (Gosselin et al 2011). A detecção qualitativa de substâncias tóxicas é bastante difundida e aceita na entomologia forense. No entanto, a análise quantitativa ainda não é bem estabelecida, uma vez que a farmacocinética, nos insetos e animais modelo, das substâncias usadas em testes, ainda é pouco conhecida, podendo levar a variações nas concentrações da substância e seus metabólitos nos insetos ou substrato alimentar (Bourel et al. 2001, Gosselin 2011, Kharbouche et al. 2008, Nolte et al. 1992, Parry et al. 2011).

2.5. CLORIDRATO DE FLUOXETINA

O cloridrato de fluoxetina, um inibidor seletivo da recaptação de serotonina (ISRS), é um antidepressivo utilizado no tratamento dos sintomas de transtorno disfórico pré-menstrual,

6 transtorno obsessivo compulsivo, depressão e bulimia nervosa, principalmente. Assim como outros ISRS, a fluoxetina tem como reações adversas o desejo suicida, agitação, convulsões, sedação, perda de apetite e, consequentemente, perda de peso. Ademais, a norfluoxetina, seu metebólito ativo, tem ação longa – devido à sua meia-vida de eliminação – e compete com enzimas hepáticas, elevando níveis de outros fármacos, inclusive antidepressivos tricíclicos, podendo fazer com que estes atinjam níveis tóxicos no organismo (Goodman e Gilman 2006). Segundo o Boletim de Farmacoepidemiologia da Agência Nacional de Vigilância Sanitária (ANVISA, 2012) sobre consumo de inibidores de apetite, as capitais Goiânia (125,97 mg em 2009 e 142,32 mg em 2010) e Vitória (152,63 mg em 2011) apresentaram maior registro de consumo per capita de cloridrato de fluoxetina. No Brasil, o consumo deste medicamento em 2009 foi de quase 3,5 toneladas, segundo o Sistema Nacional de Gerenciamento de Produtos Controlados, órgão que responde à ANVISA. Ainda, Carlini e colaboradores (2009) encontraram indícios de uso inadequado da fluoxetina em Santo André, sendo esse medicamento utilizado para fins estéticos de perda de peso e, em alguns casos, prescrito juntamente com anfetaminas anoréticas. Wilcox (1987) relatou um caso de abuso de fluoxetina, para perda de peso e controle de apetite, por uma paciente com anorexia nervosa. Após administração oral, a fluoxetina é praticamente toda absorvida e, devido ao metabolismo no fígado, possui biodisponibilidade baixa, sendo excretada quase completamente como norfluoxetina e outros metabólitos. Seus compostos ativos possuem volume de distribuição elevado, com acúmulo extensivo nos tecidos, por isso apresentam meia-vida de eliminação longa. A meia-vida de eliminação da fluoxetina é de 4 a 6 dias e da norfloxetina, de 4 a 16 dias. Ambas estão disponíveis em dois compostos cada, S-enantiômero e R-enantiômero, e os quatro compostos são ISRS, o que dificulta o estabelecimento de uma relação entre: dose administrada / concentração de fluoxetina e norfluoxetina no organismo / efeito do medicamento (Gram 1994). Ainda, a cinética da fluoxetina não é linear, as concentrações no sangue não aumentam de acordo com o aumento da dose e doses sucessivas levam a aumento na meia-vida de eliminação e biodisponibilidade. (Gram 1994, Hiemke e Härtter 2000)

3. OBJETIVOS a. Verificar o tempo de incubação dos ovos e taxa de eclosão das larvas de Chrysomya megacephala (F.) e Chrysomya putoria (W.) (Diptera: Calliphoridae) em oito faixas térmicas: 5, 10, 13, 17, 20, 25, 30 e 35 ± 1 °C; b. Verificar se as taxas de desenvolvimento dos imaturos de C. megacephala e C. putoria criadas em oito faixas térmicas: 5, 10, 13, 17, 20, 25, 30 e 35 ± 1 °C se alteram mediante a presença de cloridrato de fluoxetina em fígado de coelho; c. Reformular os modelos de graus-hora acumulados (“acumulated degree hours” - ADH) para ovos e imaturos das duas espécies a partir dos dados obtidos.

4. CAPÍTULO I –

EGG DEVELOPMENTAL TIME AND SURVIVAL OF Chrysomya megacephala (F.) AND

Chrysomya putoria (W.) (DIPTERA: CALLIPHORIDAE) UNDER DIFFERENT TEMPERATURES 1

TEMPO DE DESENVOLVIMENTO E SOBREVICÊNCIA DE OVOS DE Chrysomya megacephala (F.) E

Chrysomya putoria (W.) (DIPTERA: CALLIPHORIDAE) EM DIFERENTES TEMPERATURAS

M. A. Alonso*, C. M. Souza*, A. X. Linhares*, P. J. Thyssen*

*Department of Animal Biology, Institute of Biology, University of Campinas - UNICAMP, 255 Monteiro Lobato St., Campinas, SP, Brazil. P.O.Box 6109, P.C. 13083-862

4.1. RESUMO

Chrysomya megacephala (F.) e Chrysomya putoria (W.) (Diptera: Calliphoridae) são consideradas de importância forense, média e veterinária no Brasil, devido ao seu comportamento necrófago e sinantrópico. O desenvolvimento de moscas pode ser influenciado pela temperatura e espécies do mesmo gênero normalmente apresentam respostas diferentes para variáveis externas. O tempo de desenvolvimento dos ovos de moscas varejeiras pode ser uma técnica complementar útil para estimar o intervalo pós-morte mínimo. Assim, o objetivo desse estudo foi comparar o tempo de desenvolvimento e sobrevivência dos ovos de C. megacephala e C. putoria em diferentes temperaturas, determinar a temperatura ótima para o desenvolvimento dos ovos e a regressão linear da relação entre tempo de desenvolvimento e temperatura, determinando então o limar térmico inferior (t) e a constante térmica (K) para cada espécie. Adultos de ambas as espécies foram

1 Manuscrito escrito seguindo as normas do periódico Journal of Medical Entomology

9 coletados na região da cidade de Campinas, São Paulo, Brasil. Os ovos foram encubados em oito temperaturas constantes entre 05 ± 1 °C e 35 ± 1 °C e o tempo de desenvolvimento e a sobrevivência foram avaliados. Não houve eclosão dos ovos a 5 °C e 10 °C. Os K para C. megacephala e C. putoria foram 179.41 GH e 189.94 GH, respectivamente. O ângulo da reta da regressão linear a o t (10 °C) foram similares entre as espécies. A temperatura ótima para sobrevivência dos ovos foi entre 25 e 35 °C, para C. megacephala e entre 20 e 30 °C, para C. putoria. Os dados apresentados se assemelham à maioria dos dados disponíveis na literatura, no entanto diferenças dentro do mesmo gênero e intraespecíficas são possíveis.

5. Palavras chaves: Moscas varejeiras, Exigência térmica, Insetos necrófagos

5.1. ABSTRACT

Chrysomya megacephala (F.) and Chrysomya putoria (W.) (Diptera: Calliphoridae) are considered of forensic, medical and veterinary importance in Brazil, due to their necrophagous and synanthropic behaviour. The development of flies can be influenced by temperature and species from the same genus usually have different responses to external variables. The egg developmental time of Blow fly can be a useful complementary technique to estimate the minimum postmortem interval. Thus, this study aimed to compare the egg developmental time and survival of C. megacephala and C. putoria at different temperatures, to determine the optimal temperature for egg development and the linear regression for developmental time and temperature, and thereby determining minimum threshold (t) and thermal summation constant (K) for each species. Adults of both species were collected in the region of Campinas city, São Paulo state, Brazil. Eggs were incubated at eight constant temperatures between 05 ± 1 °C and 35 ± 1 °C and the egg developmental time and survival were evaluated. There was no egg survival at 5 °C and 10 °C. The K for C. megacephala and C. putoria were 179.41 HD and 189.94 HD, respectively. The regression slopes and t (10 °C) were similar for both species. The optimal temperature for egg survival was between 25 and 35 °C, for C. megacephala and 20 and 30 °C, for C. putoria. The present data were similar to most data available in the literature, but differences in the same genus and species are a possibility.

6. Key words: Blowflies, Development, Threshold, Necrophagous insects 10

6.1. INTRODUCTION

Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) is attracted by carcasses, of mammals and birds, and human faeces (Prins 1982) for oviposition (D´Almeida 1988). Adults of Chrysomya putoria (Wiedemann, 1830) (Diptera: Calliphoridae) are commonly found in latrines and cesspits and breeds in poultry dung (Conway 1972, Hulley 1983, Rognes and Paterson 2005). Both species can be found breeding in animal carcasses (Guimarães et al. 1978, Rognes and Paterson 2005) and have also been reported as mechanical vector of several viruses, bacteria, protozoan cysts and other enteric pathogens (Greenberg 1971, 1973; Guimarães et al. 1978), occasionally causing myiasis in traumatic lesions of animals, including humans (Zumpt 1965, Guimarães et al. 1978, Ferraz et al. 2005), and infesting foodstuff (Guimarães et al. 1978). These species were also reported colonizing corpses in the Brazilian States of Paraíba, Pernambuco, Rio de Janeiro and São Paulo (Carvalho et al. 2000, Oliveira-Costa et al. 2001, Andrade et al. 2005, Oliveira and Vasconcelos 2010). Therefore, they are considered of forensic, medical and veterinary importance in Brazil. The development of Diptera species can be influenced, for example, by temperature, relative humidity, photoperiod, and latitude (Wells and Kurahashi 1994, Mello et al. 2012, Nassu et al. 2014). Studies have also demonstrated that species of the same genus can exhibit different developmental rates even under similar rearing conditions, such as temperature and/or the presence of drugs (Lefebvre and Pasquerault 2004, Sukontason et al. 2008, Niederegger et al. 2013, Rezende et al. 2014). In the medical-legal context, the developmental parameters of flies are used mainly for calculating the post-mortem interval (PMI) (Greenberg 1991; Catts and Goff 1992). The minimum post-mortem interval (PMImin), time between the beginning of body colonization by insects and the discovery of the corpse (Catts and Goff 1992), can be calculated using linear models of development (e.g. Wagner et al. 1984, Ikemoto e Takai 2000). Developmental rates of insects at different temperatures have been studied for forensic purposes in order to improve the accuracy on the PMImin estimative (Amendt et al. 2004). Temperatures above or below the temperature threshold inherent to each species can delay the egg incubation time or disrupt, even temporarily, the development of the immature by interfering with their physiological processes (Wigglesworth 1972, Richards et al. 2009a) and, consequently, affect the egg survival (Yang and Shiao 2014). Considering that, generally, blow fly species arrive and

11 lay eggs within few minutes after the death (Catts 1992, Campobasso et al. 2001), the use of egg developmental time can be a useful complementary technique to estimate the time elapsed from the death until the discovery of the body (VanLaerhoven and Anderson 2001; Bourel et al. 2003; Tarone et al. 2007), especially in cases of early deaths (VanLaerhoven and Anderson 2001). In this way, the demand for studies of blow fly egg developmental time under different temperatures, for forensic application, is remarkable. Thus, this study aimed to compare the egg developmental time and survival of C. megacephala and C. putoria at different temperatures, to determine the optimal temperature for egg development and the linear regression for developmental time and temperature, and thereby determining minimum threshold (t) and thermal summation constant (K) for each species and to compare these parameters with the data available on the literature.

6.2. MATERIALS AND METHODS

Collection of flies and colonies establishment in the laboratory.

Adults of C. megacephala and C. putoria were collected in the metropolitan region of Campinas city (22°54'21''S, 47°03'39'' W), State of São Paulo, Brazil. Chrysomya megacephala was collected in an urban area, using chicken gizzards and rotten ground beef as baits, while C. putoria was collected in the vicinity of a poultry farm, both with the aid of an entomological net. Specimens were placed in freezer (-20 °C) for three minutes to proceed trial and identification, using an interactive taxonomic key (Grella and Thyssen 2011). Then, the species of interest were kept in plastic cages with water ad libitum, sugar and protein, at controlled temperature (25 ± 1 °C), humidity (70 ± 10%) and photoperiod (12 h), to establish colonies.

Egg developmental time development.

For the experiments, six cages of adult flies of each species were used. Four small Petri dishes without the lids, with 2 cm diameter liver beef pieces each, were put in each cage as oviposition substrate and observed every 30 minutes. The Petri dishes with an egg mass with approximately 0.5 cm of diameter were removed from the cages and inserted in larger Petri dishes with lids, to prevent hatched larvae to escape. The closed Petri dishes were placed on growth chambers (Model 202/4, Eletrolab™, São Paulo, SP) with controlled photoperiod (12 h) and constants temperatures of 5, 10, 13, 17, 20, 25, 30 and 35 ± 1 °C. This procedure was repeated until there were four

12 replicates for each species and temperature. The replicates were placed in the same growth chamber and ran simultaneously. The eggs were not manipulated to prevent any interference on the egg survival, therefore their counting were performed only after the larval hatching. The Petri dishes were also observed every 30 minutes until the beginning of larval hatching or up to 168 hours, if no larval hatching was observed. The Petri dishes without egg survival were discarded without counting the eggs.

Egg survival.

After five hours from the beginning of hatching, the Petri dishes were sealed with Parafilm M™ and stored in freezer. For counting the larvae and chorions, the Petri dishes were removed from the freezer and let untouched until they reached room temperature, then the egg masses were separated with a soft thin brush and saline solution to proceed the counting. Both the larvae that had successfully hatched and the remained eggs were counted with the aid of a stereomicroscope (Model Stemi SV 11™, Carl Zeiss, Oberkochen, BW) and the egg survival was calculated using the equation: hatched larvae / (hatched larvae + remained eggs).

Data analysis.

The ANCOVA test (PROC GLM, SAS Institute 2009) was used to compare the regression slopes of the two species, data were analysed using SAS™ (Statistical Analysis System) (SAS 2006) software with an overall error rate (α) of 0.05. Quadratic regression (Crawley 2007) was used to indicate the optimum temperature for egg survival and Mann-Whitney U-test (Crawley 2007) was used to compare the egg survival of both species in each temperature, using R Core Team (2013) system. For comparison of the data collected in this paper concerning developmental time versus temperature and the data pooled from literature, a graphic was made using ExcelTM 2013.

Linear model.

The linear model used to determine the ADH for the egg developmental time was calculated using the equations according to Ikemoto and Takai (2000) Method 2: (DT) = K + tD , that relates duration of development (D) in hours, temperature of development (T) in degrees, minimum developmental threshold (t) in degrees and thermal summation constant (K). In the figures, the lines

13 represented by this equation have x = D and y = DT. The calculus and figures were made using SAS™ (Statistical Analysis System) (SAS 2006).

6.3. RESULTS

The mean number of eggs per temperature ranged from 100 to 867 for C. megacephala, from 89 to 743 for C. putoria, and there was no hatching recorded at 5 °C and 10 °C (Table 1). The egg survival was higher between 25 °C and 35 °C for C. megacephala and between 20 °C and 30 °C for C. putoria (Figure 1) and was different between the species only at 20 °C (p=0.0294). The relation between egg developmental time and temperature did not differ between both species, according to ANCOVA test (p = 0.7813; R2 = 0.754; SD = 1.38). For both species, equations of the development were calculated assuming that the relationship between the time of development and temperature is linear. The curvature on temperatures above and below thresholds were considered, but all points were part of the linear relationship. For C. megacephala, the equation was y = 179.41 + 10.82x; R² = 0.972 (Figure 2), and, according to that, t = 10.8 °C (SE = 0.82) and K = 179.41 HD (SE = 26.69). For C. putoria, the equation was y = 189.94 + 10.29x; R² = 0.997 (Figure 2), t = 10.3 °C (SE = 0.25) and K = 189.94 HD (SE = 8.21). The egg developmental time decreased with the temperature increase, as expected, varying from over 64 h at 13 °C to seven hours at 35 °C, for C. megacephala, and, for C. putoria, between 69 h at 13 °C and eight hours at 35 °C (Figure 3). The egg developmental time for C. megacephala was similar to the data available on the literature, restricted to temperatures around 26 °C, for populations from South Africa (Prins 1982), India (Wells and Kurahashi 1994) and Brazil (Barros- Cordeiro and Pujol-Luz 2010), but diverged of a population from Egypt (Gabre et al. 2005) (Figure 3). For C. putoria, the egg developmental time was similar to the 15.5 hours presented by Greenberg and Szyska (1984), if the mean temperature of development considered is 23.9 °C (higher and lower temperatures during the development of 21.7 (± 1.9) and 26.0 (± 3.1) °C, respectively).

6.4. DISCUSSION

The thermal requirements achieved for the egg development differ from those present in the literature for the adults of C. megacephala and C. putoria, although it was expected this would not

14 vary once the metabolism kinetics tend to be constant at all insects stages (Sharpe and DeMichele 1977). Richards and colleagues (2009a) observed that the thermophysiological thresholds for the adults of C. megacephala and C. putoria were around 21 and 24 °C, respectively. An average minimum developmental threshold for adults of 10.40 °C (experimental data) and of 14.68 °C (pooled data from the literature), besides an upper critical temperature of about 35 °C (experimental data) for C. megacephala were provided by Richards and Villet (2009). For C. putoria, the minimum developmental threshold estimated by Richards et al. (2009b), considering all developmental landmarks, except egg developmental time, was of 13.42 °C, and the upper critical temperature of about 49 °C for third-instar maggots (Richards et al. 2009a). Wells and Kurahashi (1994) determined C. megacephala egg developmental time between 12 and 18 hours at 27 °C, Prins (1982) and Barros-Cordeiro and Pujol-Luz (2010) determined a duration of 14 h and 15 h, respectively, at 26 °C and Richards and Villet (2009) observed egg developmental time between 19 and 21 h for 22 °C, all somehow similar to the results here presented for 20 °C (21 h) and 25 °C (12.5 h). However, the development presented by Gabre et al. (2005), of 24 h at 26 °C for a population from Egypt, was twice of the time recorded in the other studies. The t here determined for C. megacephala egg developmental time was lower to the one estimated by Richards and Villet (2009) compilation, of 12.26 °C, as to the K = 195.8 h from their pooled data. Lefebvre and Pasquerault (2004) pointed out the importance to consider that same species can present different developmental time depending on their geographic region, due to adaptive changes triggered by environmental characteristics. For C. putoria, egg developmental time data of Greenberg and Szyska (1984) was of 14.5 and 16.5 h for two groups of eggs exposed to temperatures that fluctuated between 21.7 and 26.0 °C. This data can be similar to the one presented here at 25 °C (13 h) if the temperature of development considered is the mean temperature. Thought fluctuating temperatures might retard or speed the insects´ development (Greenberg 1991), Anderson (2000) asserted that the error caused by the use of the duration of development data under constant temperatures can be conservative for the PMImin estimate. In addition, our results showed no differences between the slopes of C. megacephala and C. putoria, indicating there is no need of doing the egg differentiation between these two species in order to use these data on the PMImin estimate based on egg development for the region of Campinas city.

The egg developmental time of C. megacephala and C. putoria decreased with the increasing of the temperature, as observed in the Greenberg and Kunich (2002) compilation for another Calliphoridae species. In the same way, the egg survival of both species was higher with the increasing of the temperature, as previous recorded for C. megacephala by Yang and Shiao (2014). The higher egg survival for C. megacephala between 25 and 35 °C and for C. putoria between 20 and 30 °C, are in accordance to the expected. Yang and Shiao (2014) obtained the highest values of C. megacephala egg survival at 20 and 25 °C. In Campinas, between 1998 and 2008, the annual average temperature was of 22.4 °C and the hotter and colder months had a difference of 6.4 °C between average temperatures (Cepagri 2015). The minimum average of July was of 12.3 °C (Cepagri 2015), when eggs of C. megacephala and C. putoria would take 64 h and 69 h to develop and only 22% and 15% of eggs would survive, respectively. While in February, the maximum average was of 30 °C (Cepagri 2015), so the C. megacephala and C. putoria egg developmental time would be of 8.5 and 8.6 h and egg survival of 80% and 83%, respectively. Sukontason et al. (2008) studied C. megacephala and C. rufifacies development under natural temperatures in Thailand (averages between 18.4 and 31.4 °C in the studied year), observing the egg developmental time of 12–24 h, suggesting the addition of 24 h in the Thailand mean temperatures for corresponding to the embryonic development. This recommendation should not be applied to PMImin estimate based on the egg developmental stage in view of our results, which pointed out that the developmental time of the egg is temperature dependent and might be known for the PMImin estimate accuracy, as stressed by VanLaerhoven and Anderson (2001). As described in Greenberg (1991) and Anderson and Cervenka (2002) case reports, the data presented for C. megacepahala and C. putoria contribute with useful information for the PMImin estimate based on the egg developmental stage for Campinas city, and improve the knowledge of natural history of these Calliphoridae species, providing new data about their biological features.

6.5. ACKNOWLEDGEMENTS

Financial support grant (#2013/07022-0) to M. A. Alonso, São Paulo Research Foundation (FAPESP).

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Nassu, M. P., P. J. Thyssen, and A. X. Linhares. 2014. Developmental rate of immatures of two fly species of forensic importance: Sarcophagidae (Liopygia) ruficornis and Microcerella halli (Diptera: Sarcophagidae). Parasitol Res 113(1): 217–222

Niederegger, S., N. Wartenberg, R. Spiess, and G. Mall. 2013. Influence of food substrates on the development of the blowflies Calliphora vicina and Calliphora vomitoria (Diptera, Calliphoridae). Parasitol Res 112(8): 2847–2853. doi:10.1007/s00436-013-3456-6

Oliveira, T. C., and S. D. Vasconcelos. 2010. Insects (Diptera) associated with cadavers at the Institute of Legal Medicine in Pernambuco, Brazil: Implications for forensic entomology. Forensic Sci Int 198: 97–102. doi: 10.1016/j.forsciint.2010.01.011

Oliveira-Costa, J., C. A. Mello-Patiu, and S. M. Lopes. 2001. Dípteros muscóides associados com cadáveres humanos na cena da morte no estado do Rio de Janeiro - Brasil. Bol Mus Nac Zoo 464: 1–6

Prins, A. J. 1982. Morphological and biological notes on six South African blow-flies. Ann S Afr Mus 90(4): 201–217

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Rezende, F., M. A. Alonso, C. M. Souza, P. J. Thyssen, and A. X. Linhares. 2014. Developmental rates of immatures of three Chrysomya species (Diptera: Calliphoridae) under the effect of methylphenidate hydrochloride, phenobarbital, and methylphenidate hydrochloride associated with phenobarbital. Parasitol Res 113(5): 1897–1907. doi:10.1007/s00436-014-3837-5

Richards, C. S., and M. H. Villet. 2009. Data quality in thermal summation development models for forensically important blowflies. Med Vet Entomol 23(3): 269–76. doi:10.1111/j.1365- 2915.2009.00819.x

Richards, C. S., B. W. Price, and M. H. Villet. 2009a. Thermal ecophysiology of seven carrion- feeding blowflies in Southern Africa. Entomol Exp Appl, 131(1): 11–19. doi:10.1111/j.1570- 7458.2009.00824.x

Richards, C. S., K. L. Crous, and M. H. Villet. 2009b. Models of development for blowfly sister species Chrysomya chloropyga and Chrysomya putoria. Med Vet Entomol 23: 56-61

Rognes, K., and H. E. H. Paterson. 2005. Chrysomya chloropyga (Wiedemann, 1818) and C. putoria (Wiedemann, 1830) (Diptera: Calliphoridae) are two different species. Afr Entomol 13(1): 49–70

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C. megacephala C. putoria Duration of Duration of Temp No. eggs Egg survival No. eggs Egg survival development development (°C) ± SD (%) ± SD ± SD (%) ± SD ± SD ± SD

5 NA NA 0 NA NA 0 10 NA NA 0 NA NA 0

13 818 ± 89 64.0 ± 1.4 22.7 ± 6.3 436 ± 343 69.0 ± 2.1 15.4 ± 8.2

17 867 ± 254 39.4 ± 8.5 22.6 ± 28.0 743 ± 437 28.4 ± 0.3 64.4 ± 20.8

20 205 ± 59 21.1 ± 0.6 66.2 ± 10.7 89 ± 63 21.0 ± 0.0 90.2 ± 7.6

25 142 ± 72 12.8 ± 0.0 84.8 ± 14.1 137 ± 86 13.0 ± 0.4 68.1 ± 19.1

30 100 ± 51 8.4 ± 0.3 80.8 ± 12.6 269 ± 161 8.6 ± 0.4 83.4 ± 12.8

35 125 ± 61 6.5 ± 0.0 82.9 ± 12.6 401 ± 545 8.0 ± 0.6 63.5 ± 17.6

NA- not applicable

Figure 2. Temperature (T) and Duration of development (D) of C. megacephala (F) and C. putoria (W.). The regression lines are used to determine t and K for egg development for each species.

Figure 3. Developmental time at different temperatures for C. megacephala (F.) and C. putoria (W.) data here presented and published data. 1- Greenberg and Szyska 1984; 2- Gabre et al. 2005; 3- Prins et al. 1982; 4- Barros-Cordeiro and Pujol-Luz 2010; 5- Wells a and Kurahashi 1994.

7. CAPÍTULO II –

EFFECT OF DIFFERENT TEMPERATURES AND PRESENCE OF FLUOXETINE HYDROCHLORIDE

ON THE DEVELOPMENT OF FORENSIC IMPORTANCE SPECIES Chrysomya megacephala (F.)

AND Chrysomya putoria (W.) (DIPTERA: CALLIPHORIDAE)2

EFEITO DE DIFERENTES TEMPERATURAS E PRESENÇA DE CLORIDRATO DE FLUOXETINA NO

DESENVOLVIMENTO DAS ESPÉCIES DE IMPORTÂNCIA FORENSE Chrysomya megacephala (F.) E

Chrysomya putoria (W.) (DIPTERA: CALLIPHORIDAE)

Marcela A. Alonso1/+, Patricia J. Thyssen1

1Department of Animal Biology, Institute of Biology, P.O.Box 6109, University of Campinas - UNICAMP, 13083-862, Campinas, SP, Brazil +Corresponding author: [email protected], +55 19 996457212

7.1. ABSTRACT

Calliphoridae (Insecta: Diptera) tem importância forense em muitos países por ser frequentemente utilizada na estimativa do intervalo pós-morte (IPM). Para o cálculo do intervalo pós-morte mínimo

(IPMmin) o conhecimento acerca do desenvolvimento dos insetos sob condições bióticas e abióticas variadas é imprescindível. E estudo objetivou avaliar o desenvolvimento de Chrysomya megacephala (F.) e Chrysomya putoria (F.) (Diptera: Calliphoridae) sob diferentes temperaturas (13, 17, 20, 25, 30, 35 °C) combinadas ou não com a ação de fluoxetina. O desenvolvimento das duas espécies foi diferente para a interação entre fluoxetina e temperatura, considerando peso e comprimento como as variáveis respostas (p < 0,05). Os resultados mostram que o IPMmin dessas

2 Manuscrito escrito seguindo as normas do periódico Forensic Science International 26 espécies pode ser subestimado em 24h a 17 °C ou superestimado em 12h a 35 °C, se a interação entre as duas variáveis não for considerada. Estudos considerando a presença de outras drogas simultaneamente com diferentes temperaturas devem ser realizados para aumentar o conhecimento acerca das variáveis que podem afetar o desenvolvimento de espécies necrófagas e, consequentemente, a estimativa do IPMmin.

Palavras-chave: Entomologia forense, Entomotoxicologia, Intervalo pós-morte, Inseto necrófago.

7.2. RESUMO

Calliphoridae (Insecta: Diptera) is of forensic importance in many countries for being frequently used for the post-mortem interval (PMI) estimate. For the minimum post-mortem interval (PMImin) calculus is important to know the insect development under various biotic and abiotic conditions. Thus, this study aimed to evaluate the development of Chrysomya megacephala (F.) and Chrysomya putoria (W.) (Diptera: Calliphoridae) at different temperatures (13, 17, 20, 25, 30 and 35 °C) with and without fluoxetine hydrochloride on the rabbit liver used as rearing substrate. The development of both species was different for the fluoxetine hydrochloride and temperature interaction in relation to control group, considering weight and body length as the response variables (p < 0.05). Results showed that the PMImin based on those species development could be under estimated in 24h at 17 °C or overestimated in 12h at 35 °C if the interaction between both variables is not considered. Further research with other drugs presence and different temperatures as simultaneous variables must be performed to increase the knowledge about factors that might affect the scavenger species development and, consequently, the PMImin estimate.

Key words: Forensic entomology, Entomotoxicology, Blowflies, Post-mortem interval, Scavenger

7.3. INTRODUCTION

Calliphoridae are well known for being the first dipterans to reach a cadaver and for their great abundance, especially in tropical regions (Carvalho and Linhares 2001). Originally with an Australasian and Pacific distribution, Chrysomya megacephala (Fabricius 1794) (Diptera: Calliphoridae), also known as oriental latrine fly, is now common in New World (Guimarães et al. 1978, Wells 1991) and Chrysomya putoria (Wiedemann 1830) (Diptera: Calliphoridae), also an introduced species in South America, is now widespread in this continent (Greenberg and Kunich 2002). Both species are reported as vectors of pathogens (Wells 1991) and of forensic importance (Carvalho et al. 2000). Therefore, the studies about blowflies’ lifecycles, behaviour and distribution are of major importance to improve the accuracy of their use as forensic indicators of minimum post-mortem interval (PMImin). The PMImin is the time elapsed between the beginning of the colonization of a corpse and its discovery, and it can be estimated based on the necrophagous insect age (Catts and Goff 1992). The insects development is influenced by biotic and abiotic factors such as temperature, rearing substrate composition and presence of drugs in the substrate (Wigglesworth 1972, Goff and Lord 1994). The effect of the temperature on forensic important insects is one of the most studied variables. In most cases, low temperatures increase the total developmental time of insects and high temperatures decrease it (Campobasso et al. 2001). However, different populations of the same species may have distinct behavior and responses to variations on the ambient, due to genetic and environmental factors, so it is important to have knowledge about multiples populations to improve the PMImin estimate accuracy (Gallagher et al. 2010). In addition, for the accumulated degree hour

(ADH) or day (ADD) calculus, in which the PMImin is based, is crucial to know the minimum and / or maximum temperatures thresholds, what increases the importance of insect development in different temperatures studies (Amendt et al. 2004). Entomotoxicology, a branch of forensic entomology, aims to evaluate if there was drug use before death, especially when there is no sample with suitable conditions on the corpse for toxicological analysis (Beyer et al. 1980, Bourel et al. 1999, Introna et al. 2001). In addition, there is interest in evaluate if the drug somehow influences the insect physiology (Introna et al. 2001) or behaviour, which may lead to errors on the PMI estimate (Ullyett 1950, Hanski 1987, Greenberg and Kunich 2002). Lü and colleagues (2014) observed a delay on the development of immature of

C. megacephala reared in the presence of ketamine in three different temperatures, and Thyssen and colleagues (2011) also reported a delay on the development of C. putoria under effect of scopolamine. The fluoxetine hydrochloride is a selective serotonin reuptake inhibitor commonly prescribed for the symptoms of premenstrual dysphoric disorder, obsessive-compulsive disorder, depression and bulimia nervosa (Gram 1994). This medicine also has as adverse reactions, among others, suicidal thoughts, agitation, convulsions, sedation and appetite loss, being used for weight loss in some cases (Wise 1992). The fluoxetine half-life of excretion is between four and six days and of norfluoxetine, its primary metabolite, is from seven to 15 days and they both are potent inhibitors of the reuptake of serotonine (Gram 1994). This study aimed to evaluate the development of larvae of C. megacephala (F.) and C. putoria (W.) (Diptera: Calliphoridae) reared on animal tissue with fluoxetine hydrochloride at different temperatures, thereby determining larval minimum threshold and thermal constant for each species.

7.4. MATERIALS AND METHODS

Male New Zealand White rabbits (Oryctolagus cuniculus, Linnaeus 1758 (Lagomorpha: Leporidae)), with approximately four kilos each, were kept in individual cages under normal laboratory conditions (natural temperature and day/night cycle) and free access to food and water on the Centre of Experimental Medicine and Surgery for three days before the beginning of the experiments. Fluoxetine hydrochloride (Daforin®, EMS, oral solution 20 mg/ml) was administrated to six rabbits via oral gavage at 9:00 am during four days. The medicine was diluted in distillate water, in doses equal to 1 mg per kilo on the first two days and 3 mg per kilo on the last two days. The six animals from the control group received 10 ml of saline solution during four days. At 2:00 pm of the fourth day the rabbits were euthanized via CO2 asphyxiation and had their livers removed immediately. Each liver, weighting approximately 200 g, was divided in two portions that were stored on different plastic vials with sawdust for larvae to crawl into and reach pupae stage after feeding, both vials were kept at the same temperature. The procedures were authorized by Commission of Ethics on the Use of Animals, protocol number 3274-1. The larvae were obtained from adults of C. megacephala collected in the urban area and adults of C. putoria collected in a poultry farm near Campinas city, both in the State of São Paulo, 29

Brazil. Both colonies were kept in plastic cages with water ad libitum and a mixture of sugar, brewer's yeast and powder milk, in controlled temperature (25 ± 1 °C), humidity (70 ± 10%) and photoperiod (12 h). In order to stimulate the ovarian development, raw liver beef was offered every three days after adults emergence until the beginning of the experiments. Raw liver beef was also used as oviposition substrate. Newly hatched larvae, from 7th laboratory generation, were placed over the rabbit livers portions in a proportion of 1.5 larvae per 1 g of tissue. The experimental vials were kept in environmental chambers, EletrolabTM model 202/4, with the controlled temperatures: 13, 17, 20, 25, 30 and 35 ± 1 °C and 12h photophase. For the immature developmental curves, five larvae were individually weighted every 12h until pupae stage, killed in hot water (± 70 ºC), fixed in Kahle´s solution (30 mL of ethanol 95%, 12 mL of formaldehyde, 4 mL of glacial acetic acid and 60 mL of distilled water) and kept at room temperature (approximately 25 °C) for posterior body length measures. At the beginning of development, the five larvae were weighted in together until they reached minimum weight of 0.0020 g, due to variations on the scale. The weights were taken using a precision scale 0.0001 g (Bel engineeringTM) and the body length was measured with the help of a stereomicroscope ZeissTM Discovery V.12 and image capture system AxioCam 5.0TM and software ZENTM version 2.0 (Figure 3).

Figure 4. Example of Chrysomya megacephala (F.) (Diptera: Calliphoridae) body length measurement with stereomicroscope and image capture system.

The mean and standard errors were determined for weight and body length. One-way ANOVA and Duncan multiple comparisons test (PROC GLM, SAS Institute 2009) were used to determine differences or similarities between the means of fluoxetine hydrochloride and temperatures groups and the control groups. The data were analysed using SAS™ (Statistical Analysis System) (SAS 2006) software with an overall error rate (α) of 0.05. The linear model of the larval development with and without fluoxetine hydrochloride on the rearing substrate was calculated using Ikemoto and Takai (2000) model 2. From this regression the thermal summation constant (K) and minimum threshold (t) were determined in ADD. Microsoft Excel™ 2013 was used to prepare the graphics.

7.5. RESULTS AND DISCUSSION

The fluoxetine hydrochloride and temperature together had a significant influence over the species, in relation to control group: for C. megacephala weight p = 0.0025 and body length p < 0.0001 and for C. putoria weight p < 0.0001 and body length p < 0.0001. As expected, for both species, the larval developmental time decreased with the temperature raising, from 254 h at 17°C to 74 h at 35 °C for control and from 278 h to 86 h for fluoxetine hydrochloride group (Figures 5 to 10). At 13 °C all the larvae died before reaching the pupal stage (Figures 5 and 8), which could be expected due to tropical distribution of the species (Zumpt 1965) For C. megacephala, the development slowed down in the presence of fluoxetine hydrochloride at the lowest temperature (17 °C) (Figure 5), but was faster from 25 to 35 °C (Figures 6 and 7), though the only temperature with an statistical different was at 30 °C, according to Chi- square test (χ2 = 7.04; p = 0.0080). For C. putoria, the development also slowed down with the fluoxetine hydrochloride at 17 °C (Figure 8), and was faster at 35 °C (Figure 10). The Lü and collaborators (2014) study with ketamine and different temperatures showed that the influence of the drug also varied with temperature and the group reared in the lowest temperature (24 °C), but not with the lowest ketamine concentration, was the one with the highest suppressed development. The Duncan multiple comparisons test for the analysis of temperature effect on the development inside the control or fluoxetine hydrochloride groups presented differences between the response variables. For C. megacephala, on the control group, the mean weight of the temperatures 17 and 20 °C was different, but the body length was not, and on the fluoxetine group, mean body lengths of 20, 25 and 35 °C were different, while the weight was not (Table 2). For C. 31 putoria, the temperatures 30 and 35 °C presented differences on the mean weight but not on the body length (Table 2). The differences between the response variables were also detected by Duncan multiple comparisons test on the analysis of the fluoxetine hydrochloride effect in each temperature. For C. megacephala, at 25 °C the weight means were different between the groups, but the body length means were not, and at 35 °C the body length was different between control group and fluoxetine hydrochloride group, but the weight was not (Table 2). And, although for C. putoria both means were statistically different between the groups at 17 and 20 °C, at 35 °C the difference was only detected for weight means (Table 2). This result could be due different larval response to hot water and fixation in Kahle’s solution for body length measure, since not all larvae died with their body muscles fully relaxed, even if the procedure is the same and they were killed at the same time. The difference among individuals, as larval weight and total amount of fat tissue in their body might interfere on the killing and fixation process, although according to Greenberg and Kunich (2002), killing immature in hot water would guarantee fully body extension regardless the preservative liquid. Also, Lü and collaborators (2014) had similar results about differences about body length and weight, suggesting that the relation between this two measures are not linear. The developmental linear regressions for both species were different for control and fluoxetine hydrochloride groups. For both species, the minimum threshold (t) was higher for the group with fluoxetine hydrochloride on the rearing substrate and the thermal summation (K) was smaller. For C. megacephala control group, the development equation is: y = 98.97 + 7.28x (R2 = 0.91), and thermal parameters K = 99 DD (SE = 7.82) and t = 7.3 °C (SE = 1.16) (Figure 6), and for fluoxetine hydrochloride group is: y = 65.93 + 11.21x (R2 = 0.97), and being K = 66 DD (SE = 6.78) and t = 11.2 °C (SE = 1.0) (Figure 11). For C. putoria control group, the development equation is: y = 80.36 + 9.15x (R2 = 0.82), then K = 80 DD (SE = 13.13) and t = 9.2 °C (SE = 2.03) (Figure 6), and for fluoxetine hydrochloride group is: y = 69.63 + 10.77x (R2 = 0.94), thereby K = 70 DD (SE = 9.08) and t = 10.8 °C (SE = 1.34) (Figure 12). The results, for C. megacephala fluoxetine hydrochloride group, were similar to the ones presented by Richards and Villet (2009), for minimum temperature, for experimental data, between 10.57 and 12.49 °C, but not for pooled data, between 16.49 and 19.32 °C, for larval development. However, the thermal summation constants were different from both experimental (K = 150.61 DD) and pooled data (K = 44.19 DD) (Richards and Villet 2009). In addition, they suggested a critical development temperature between 17 and 33 °C for this species. For C. putoria, the data

32 from Richards and colleagues (2009) was similar, for the control group, for thermal summation constant (K = 82.74 DD), but the minimum temperature was between 12.52 and 13.29 °C, even higher than the one from fluoxetine group. These results reinforces the importance of considering that different populations of the same species might present distinct responses to changes on the environment, due to adaptive changes, as Lefebvre and Pasquerault (2004) indicated.

7 4 A 6

3 5

4 2

Weight Weight (mg) Length Length (mm) 2 1

0 0 14 38 62 86 110 134 158 182 206 230 254 278 Age (h)

Mean length Control Mean length Fluoxetine All larvae were dead Mean weigth Control Mean weight Fluoxetine

18 70 B 16 60 14 50 12

10 40

8 30 Weigth Weigth (mg) Length Length (mm) 6 20 4 10 2

0 0 14 38 62 86 110 134 158 182 206 230 254 278 302 Age (h) Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

Figure 5. Chrysomya megacephala (F.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were A = 13 ±1 °C and B = 17 ±1 °C. All larvae died at 13 °C before reach minimum weight (0.002 g), therefore there is no SD for the temperature. Data analysis with an overall error rate (α) of 0.05.

20 90 C 18 80

16 70 14 60 12 50 10 40

Weigth Weigth (mg) Length Length (mm) 30 6

4 20

2 10

0 0 14 26 38 50 62 74 86 98 110 122 134 146 158 170 182 Age (h)

Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

25 100 D

20 80

15 60

10 40

Weigth Weigth (mg) Length Length (mm)

5 20

0 0 14 26 38 50 62 74 86 98 110 122 134 146 Age (h)

Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

Figure 6. Chrysomya megacephala (F.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were C = 20 ± 1 °C and D = 25 ±1 °C. Data analysis with an overall error rate (α) of 0.05.

20 90 E 18 80

16 70 14 60 12 50 10 40

Weigth Weigth (mg) Length Length (mm) 30 6

4 20

2 10

0 0 14 26 38 50 62 74 86 98 110 122 Age (h)

Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

20 80 F 18 70 16 60 14 50 12

10 40

8 Weigth Weigth (mg) Length Length (mm) 30 6 20 4 10 2

0 0 14 26 38 50 62 74 86 98 Age (h)

Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

Figure 7. Chrysomya megacephala (F.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were E = 30 ± 1 °C and F = 35 ±1 °C. Data analysis with an overall error rate (α) of 0.05.

9 7 G 8 6 7 5 6

5 4

4 3

Weigth Weigth (mg) Length Length (mm) 3 2 2 1 1

0 0 14 38 62 86 110 134 158 182 206 230 254 278 302 326 Age (h) Mean length Control Mean length Fluoxetine All larvae were dead Mean weigth Control Mean weight Fluoxetine

18 60 H 16 50 14

12 40

10 30

Weigth Weigth (mg) Length Length (mm) 6 20

4 10 2

0 0 14 38 62 86 110 134 158 182 206 230 254 278 302 Age (h) Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

18 70 I 16 60 14 50 12

10 40

8 30

Weigth Weigth (mg) Length Length (mm) 6 20 4 10 2

0 0 14 26 38 50 62 74 86 98 110 122 134 146 158 170 182 Age (h) Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

18 80 J 16 70

14 60 12 50 10 40

8 Weigth Weigth (mg) Length Length (mm) 30 6 20 4

2 10

0 0 14 26 38 50 62 74 86 98 110 Age (h) Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

Figure 9. Chrysomya putoria (W.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were I = 30 ±1 °C and J = 35 ±1 °C. Data analysis with an overall error rate (α) of 0.05. 38

20 70 L 18 60 16

14 50

12 40 10 30

Weigth Weigth (mg) Length Length (mm)

6 20 4 10 2

0 0 14 26 38 50 62 74 86 98 110 Age (h)

Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

18 70

16 M 60 14 50 12

10 40

8 30

Weigth Weigth (mg) Length Length (mm) 6 20 4 10 2

0 0 14 26 38 50 62 74 86 98 Age (h)

Mean length Control Mean length Fluoxetine Mean weigth Control Mean weight Fluoxetine

Figure 10. Chrysomya putoria (W.) (Diptera: Calliphoridae) development under different temperatures and with fluoxetine hydrochloride on the rearing substrate (rabbit liver), represented by weight and body length. The temperatures of development were L = 30 ±1 °C and M = 35 ±1 °C. Data analysis with an overall error rate (α) of 0.05. 39

C. megacephala C. putoria

Control group Fluoxetine hydrochloride group Control group Fluoxetine hydrochloride group Temp. Weight Body length Weight Body length Weight Body length Temp. Weight (°C) (mg) (mm) (mg) (mm) (mg) (mm) (°C) (mg) 13 0.80 f 3.48 e 1.47 d 3.93 e 0.71 f 3.33 e 1.87 e 4.36 e (NA) (NA) (NA) (NA) (NA) (NA) (NA) (NA) 17 27.03 e /A 10.68 d /A 25.84 c /A 10.43 d /A 16.09 e /A 8.39 d /A 24.00 d /B 9.85 d /B (66.9) (16.3) (64.7) (16.1) (49.9) (15.8) (54.4) (15.6) 20 30.34 d /A 10.82 d /A 31.23 b /A 11.13 c /A 29.49 d /A 10.81 c /A 27.40 c /B 10.37 c /B (71.4) (16.9) (78.6) (17.7) (56.8) (16.1) (65.7) (16.8) 25 38.36 b /A 12.42 b /A 34.12 b /B 12.03 b /A 32.2 c /A 11.80 b /A 31.13 b /A 11.41 b /A (81.3) (18.6) (82.0) (17.6) (66.2) (17.4) (57.7) (16.5) 30 53.22 a /A 14.67 a /A 48.11 a /A 14.16 a /A 37.1 b /A 12.49 a /A 38.98 a /A 12.83 a /A (84.7) (17.7) (75.7) (18.6) (57.5) (15.9) (55.8) (19.5) 35 34.32 c /A 11.51 c /A 32.94 b /A 10.61 d /B 42.18 a /A 12.47 a /A 40.18 a /B 12.58 a /A (66.9) (17.2) (67.7) (16.7) (68.3) (15.3) (63.6) (16.3) In parenthesis, maximum weight and body length during development NA- not applicable

Figure 11. Chrysomya megacephala (F.) (Diptera: Calliphoridae) developmental rate linear model (ADH) for the control (dots) and fluoxetine hydrochloride groups (crosses). The regression lines are used to determine t and K for egg development for each group. Confidence interval lines of 95%.

Figure 12. Chrysomya putoria (W.) (Diptera: Calliphoridae) developmental rate linear model (ADH) for the control (dots) fluoxetine hydrochloride groups (crosses). The regression lines are used to determine t and K for egg development for each species. Confidence interval lines of 95%.

7.6. CONCLUSIONS

The results are important to the PMImin estimative using flies once it could be under estimated in 24 h at 17 °C or overestimated in 12 h at 35 °C for C. megacephala and C. putoria, if the presence of fluoxetine hydrochloride is not considered. The drug presence also increases the minimum threshold, influencing on the ADD, used for the PMImin estimative. Thought, developmental changes of those blowflies under higher concentrations of fluoxetine hydrochloride and at different temperatures are still unknown. More researches dealing with these two variables (drug presence and temperature) should be performed in order to increase the knowledge about those species life cycle under various circumstances and, consequently, improve the PMImin accuracy.

7.7. ACKNOWLEDGEMENTS

This study was possible due to financial support grant (#2013/07022-0) to M. A. Alonso, São Paulo Research Foundation (FAPESP). We thank the help of the Nucleus of Experimental Surgery and Medicine with animal care and experimentation and the Commission of Ethics on the Use of Animals for approving our protocol.

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8. CONCLUSÕES GERAIS

O desenvolvimento dos imaturos, considerando a fase embrionária e pós-embrionária, de C. megacephala e C. putoria foi influenciado pela temperatura. Altas temperaturas favorecem a eclosão das larvas e diminuem o tempo de incubação dos ovos. A 13 °C, as larvas eclodem, mas não completam seu desenvolvimento. As variações observadas quanto ao tempo de desenvolvimento dos imaturos, considerando a fase pós-embrionária, mostram que pode haver subestimativas ou superestimavas do IPM dependendo do tipo de interação existente entre temperatura e presença de fluoxetina. A 17 °C, a interação ente fluoxetina e baixa temperatura retardou o desenvolvimento das larvas, no entanto, a 35 °C, a interação entre as duas variáveis acelerou o processo. Isso ressalta a importância de estudos que reúnam mais de uma variável combinada, quer seja biótica ou abiótica, que possam influenciar o desenvolvimento dos insetos de interesse forense, a fim de aumentar a acurácia ou evitar erros na estimativa do IPM. É valido ressaltar também que espécies do mesmo gênero e mesmo populações da mesma espécie provenientes de distintas localizações geográficas podem apresentar comportamentos e respostas diferentes aos estímulos do ambiente. Assim, os estudos em diferentes regiões geográficas, além de contribuírem para maior precisão das estimativas de interesse forense, promoveriam a ampliação de conhecimento acerca da história natural das espécies de moscas necrófagas.

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10. ANEXO