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

ii

iii

iv

v

vi

vii

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

viii

ix

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 post-

mortem 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

x

xi

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

xii

5.6. CONCLUSIONS ............................................................................................................................. 43

5.7. ACKNOWLEDGEMENTS ............................................................................................................... 43

5.8. REFERENCES ............................................................................................................................... 43

6. CONCLUSÕES GERAIS ........................................................................................................................ 46

7. REFERÊNCIAS BIBLIOGRÁFICAS ....................................................................................................... 47

8. ANEXO ................................................................................................................................................ 55

xiii

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.

xiv

xv

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

xvi

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

xvii

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

xviii

1

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

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

3

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).

4

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

5

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

6

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,

7

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)

8

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.

9

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

10

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

11

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

12

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

13

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

14

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

15

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.

16

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).

17

6.6. REFERENCES CITED

Amendt, J., R. Krettek, and R. Zehner. 2004. Forensic entomology. Naturwissenshaften 91: 51–65

Anderson, G. S. 2000. Minimum and maximum development rates of some forensically important

Calliphoridae (Diptera). J Forensic Sci 45(4): 824–832

Anderson, G. S., and V. J. Cervenka. 2002. Insects associated with the body: their use and analyses.

In: Haglund, W. D., Sorg, M. eds. Advances in Forensic Taphonomy Method, Theory and

Archeological Perspectives. CRC, Boca Raton, FL

Andrade, H. T. A., A. A. Varela-Freire, M. J. A. Batista, and J. F. Medeiros. 2005. Calliphoridae

(Diptera) coletados em cadáveres humanos no Rio Grande do Norte. Neotrop Entomol 34(5):

855–856

Barros-Cordeiro, K. B., and J. R. Pujol-Luz. 2010. Morfologia e duração do desenvolvimento pós-

embrionário de Chrysomya megacephala (Diptera: Calliphoridae) em condições de laboratório.

Papéis Avulsos de Zoologia 50(47): 709-717

Bourel, B., B. Callet, V. Hédouin, and D. Gosset. 2003. Flies eggs: a new method for the estimation

of short-term post-mortem interval? Forensic Sci Int 135(1): 27–34. doi:10.1016/S0379-

0738(03)00157-9

Campobasso, C. P., G. D. Vella, and F. Introna. 2001. Factors affecting decomposition and Diptera

colonization. Forensic Sci Int 120: 18–27

Carvalho, L. M. L., P. J. Thyssen, A. X. Linhares, and F. A. B. Palhares. 2000. A checklist of

arthropods associated with pig carrion and human corpses in Southeastern Brazil. Mem Inst

Oswaldo Cruz 95(1): 135–138

Catts, E. P. 1992. Problems in Estimating the Postmortem Interval. J Agric Entomol 9(4): 245–255

Catts, E. P., and M. L. Goff. 1992. Forensic entomology in criminal investigations. Ann Rev

Entomol 37: 253–272

18

Cepagri 2015. Centro de Pesquisas Meteorológicas e Climáticas Aplicadas à Agricultura.

http://www.cepagri.unicamp.br/outras-informacoes/clima-de-campinas.html Accessed 24 April

2015

Conway J.A. 1972. The control of blowflies (Diptera, Calliphoridae) attacking green hides in the

Gambia. Trop Anim Hlth Prod 4(2): 113–119

Crawley, M. J. 2007. The R book. John Wiley & Sons, Ltd. Chichester, England.

D´Almeida, J. M. 1988. Substratos utilizados para a criação de dípteros de caliptratos em uma área

urbana do município do Rio de Janeiro. Mem Inst Oswaldo Cruz 83(2): 201-206

Ferraz, A. C. P., B. Proença, B. Q. Gadelha, L. M. Faria, M. G. M. Barbalho, V. M. Aguiar-Coelho,

and C. S. S. Lessa. 2010. First record of human myiasis caused by association of the species

Chrysomya megacephala (Diptera: Calliphoridae), Sarcophaga (Liopygia) ruficornis (Diptera:

Sarcophagidae), and Musca domestica (Diptera: Muscidae). J Med Entomol 47(3): 487–490.

doi: 10.1603/ME09143

Gabre, R. M., F. K. Adham, H. Chi. 2005. Life table of Chrysomya megacephala (Fabricius)

(Diptera: Calliphoridae). Acta Oecol 27: 179-183

Greenberg, B. 1971. Flies and disease, vol. 1. Ecology, classification and biotic association.

Princeton University Press, Princeton, NJ

Greenberg, B. 1973. Flies and disease, vol. 2. Biology and disease transmission. Princeton

University Press, Princeton, NJ

Greenberg, B. 1991. Flies as forensic indicators. J Med Entomol, 28: 565–577

Greenberg, B., and J. C. Kunich. 2002. Entomology and the law: flies as forensic indicators.

Cambridge Universit Press, Cambridge

Greenberg, B., and Szyska, M. L. 1984. Immature stages and biology of fifteen species of Peruvian

Calliphoridae (Diptera). Ann Entomol Soc Am 77: 488-517

19

Grella, M. D., and P. J. Thyssen. 2011. Chave taxonômica interativa para espécies de dípteros

califorídeos (Infraordem: Muscomorpha) do Brasil.

http://keys.lucidcentral.org/keys/v3/calliphoridae_brazil. Accessed 10 July 2013

Guimarães, J. H., A. P. Prado, and A. X. Linhares. 1978. Three newly introduced blowfly species

in southern Brazil (Diptera, Calliphoridae). Rev Bras Entomol 22(1): 53–60

Hulley, P. E. 1983. A survey of the flies breeding in poultry manure, and their potential enemies. J

Entomol Soc Sth Afr 46(1): 37–47

Ikemoto T., and J. Takai. 2000. A new linearized formula for the law of total effective temperature

and the evaluation of line-fitting methods with both variables subject to error. Env Entomol

29(4): 671–682

Lefebvre, F., and T. Pasquerault. 2004. Temperature-dependent development of Ophyra aenescens

(Wiedemann, 1830) and Ophyra capensis (Wiedemann, 1818) (Diptera, Muscidae). Forensic

Sci Int 139(1): 75–79. doi:10.1016/j.forsciint.2003.10.014

Mello, R. S., G. E. M. Borja, and M. M. C. Queiroz. 2012. How photoperiods affect the immature

development of forensically important blowfly species Chrysomya albiceps (Calliphoridae).

Parasitol Res 111: 1067–1073

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

20

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

R Core Team 2013. R: A language and environment for statistical computing. R Foundation for

Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0. http://www.R-project.org/

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

SAS Inc. (2006) SAS for Microsoft Windows Professional, version 9.1, Cary, NC

SAS Inc. (2009) SAS/STAT(R) 9.2 User's Guide, Second Edition, Cary, NC.

http://support.sas.com/documentation/cdl/en/statug/63033/HTML/default/viewer.htm#titlepag

e.htm Accessed 20 October 2014

21

Sharpe, P. J. H. and D. W. DeMichele. 1977. Reaction kinetics of poikilotherm development. J

Theor Biol 64: 649–670

Sukontason, K., S. Piangjai, S. Siriwattanarungsee, and K. L. Sukontason. 2008. Morphology and

developmental rate of blowflies Chrysomya megacephala and Chrysomya rufifacies in

Thailand: application in forensic entomology. Parasitol Res 102(6): 1207–1216.

doi:10.1007/s00436-008-0895-6

Tarone, M. A., Jennings, K. C. and Foran, D. R. 2007. Aging blow fly eggs using gene expression:

a feasibility study. J Forensic Sci. 52(6):1350-1354 doi: 10.1111/j.1556-4029.2007.00587.x

VanLaerhoven, S. L., and G. S. Anderson. 2001. Implications of using development rates of blow

fly (Diptera: Calliphoridae) eggs to determine postmortem interval. J Entomol Soc Brit

Columbia 98: 189–194

Wagner, T. L., H. Wu, P. J. H. Sharpe, R. M. Schoolfield, and R. N. Coulson. 1984. Modeling

insect development rates: a literature review and application of a biophysical model. Ann

Entomol Soc Am 77: 208–225

Wells, J. D., and H. Kurahashi. 1994. Chrysomya megacephala (Fabricius) (Diptera:

Calliphoridae) development: rate, variation and implications for forensic entomology. Japanese

J Sanitary Zool 45(4): 303–309

Wigglesworth, V. B. 1972. The principles of insect physiology. Chapman and Hall, London

Yang, S. T., and S. F. Shiao. 2014. Temperature adaptation in Chrysomya megacephala and

Chrysomya pinguis, two blow fly species of forensic significance Entomol Exp Appl 152: 100–

107. doi: 10.1111/eea.12206

Zumpt, F. 1965. Myiasis in Man and Animals in the Old World: a Textbook for Physicians,

Veterinarians and Zoologists. Butterworth, London

22

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.

C. megacephala C. putoria

Temp

(°C)

No. eggs

± SD

Duration of

development

± SD

Egg survival

(%) ± SD

No. eggs

± SD

Duration of

development

± SD

Egg survival

(%) ± 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

23

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.

24

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.

25

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.

26

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: marcela.aq.alonso@gmail.com, +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

27

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

28

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

29

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,

30

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.

31

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.

32

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

33

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.

34

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.

0

1

2

3

4

0

1

2

3

4

5

6

7

14 38 62 86 110 134 158 182 206 230 254 278

Wei

ght

(mg)

Len

gth

(m

m)

Age (h)

A

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

All larvae were dead

0

10

20

30

40

50

60

70

0

2

4

6

8

10

12

14

16

18

14 38 62 86 110 134 158 182 206 230 254 278 302

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

B

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

35

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.

0

10

20

30

40

50

60

70

80

90

0

2

4

6

8

10

12

14

16

18

20

14 26 38 50 62 74 86 98 110 122 134 146 158 170 182

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

C

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

0

20

40

60

80

100

0

5

10

15

20

25

14 26 38 50 62 74 86 98 110 122 134 146

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

D

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

36

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.

0

10

20

30

40

50

60

70

80

90

0

2

4

6

8

10

12

14

16

18

20

14 26 38 50 62 74 86 98 110 122

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

E

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

0

10

20

30

40

50

60

70

80

0

2

4

6

8

10

12

14

16

18

20

14 26 38 50 62 74 86 98

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

F

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

37

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.

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

8

9

14 38 62 86 110 134 158 182 206 230 254 278 302 326

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

G

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

All larvae were dead

0

10

20

30

40

50

60

0

2

4

6

8

10

12

14

16

18

14 38 62 86 110 134 158 182 206 230 254 278 302

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

H

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

38

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.

0

10

20

30

40

50

60

70

0

2

4

6

8

10

12

14

16

18

14 26 38 50 62 74 86 98 110 122 134 146 158 170 182

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

I

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

0

10

20

30

40

50

60

70

80

0

2

4

6

8

10

12

14

16

18

14 26 38 50 62 74 86 98 110

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

J

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

39

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.

0

10

20

30

40

50

60

70

0

2

4

6

8

10

12

14

16

18

20

14 26 38 50 62 74 86 98 110

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

L

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

0

10

20

30

40

50

60

70

0

2

4

6

8

10

12

14

16

18

14 26 38 50 62 74 86 98

Wei

gth

(m

g)

Len

gth

(m

m)

Age (h)

M

Mean length Control Mean length Fluoxetine

Mean weigth Control Mean weight Fluoxetine

40

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.

C. megacephala C. putoria

Control group Fluoxetine hydrochloride group Control group Fluoxetine hydrochloride group

Temp.

(°C)

Weight

(mg)

Body length

(mm)

Weight

(mg)

Body length

(mm)

Weight

(mg)

Body length

(mm)

Temp.

(°C)

Weight

(mg)

13 0.80 f

(NA)

3.48 e

(NA)

1.47 d

(NA)

3.93 e

(NA)

0.71 f

(NA)

3.33 e

(NA)

1.87 e

(NA)

4.36 e

(NA)

17 27.03 e /A

(66.9)

10.68 d /A

(16.3)

25.84 c /A

(64.7)

10.43 d /A

(16.1)

16.09 e /A

(49.9)

8.39 d /A

(15.8)

24.00 d /B

(54.4)

9.85 d /B

(15.6)

20 30.34 d /A

(71.4)

10.82 d /A

(16.9)

31.23 b /A

(78.6)

11.13 c /A

(17.7)

29.49 d /A

(56.8)

10.81 c /A

(16.1)

27.40 c /B

(65.7)

10.37 c /B

(16.8)

25 38.36 b /A

(81.3)

12.42 b /A

(18.6)

34.12 b /B

(82.0)

12.03 b /A

(17.6)

32.2 c /A

(66.2)

11.80 b /A

(17.4)

31.13 b /A

(57.7)

11.41 b /A

(16.5)

30 53.22 a /A

(84.7)

14.67 a /A

(17.7)

48.11 a /A

(75.7)

14.16 a /A

(18.6)

37.1 b /A

(57.5)

12.49 a /A

(15.9)

38.98 a /A

(55.8)

12.83 a /A

(19.5)

35 34.32 c /A

(66.9)

11.51 c /A

(17.2)

32.94 b /A

(67.7)

10.61 d /B

(16.7)

42.18 a /A

(68.3)

12.47 a /A

(15.3)

40.18 a /B

(63.6)

12.58 a /A

(16.3)

In parenthesis, maximum weight and body length during development

NA- not applicable

41

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%.

42

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%.

43

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.

7.8. REFERENCES

Amendt J, Krettek R, Zehner R (2004) Forensic entomology. Naturwissenshaften 91: 51-65

Beyer JC, Enos WF, Stajic M (1980) Drug identification through analysis of maggots. J Forensic

Sci 25: 411-412

Bourel B, Hédouin V, Martin-Bouyer L, Bécart A, Tournel G, Deveaux M, Gosset D (1999) Effects

of morphine in decomposing bodies on the development of Lucilia sericata (Diptera:

Calliphoridae). J Forensic Sci 44(2): 354–358

Campobasso CP, Vella GD, Introna F (2001) Factors affecting decomposition and Diptera

colonization. Forensic Sci Int 120: 18-27

Carvalho LM, Thyssen PJ, Linhares AX, Palhares FA (2000) A checklist of arthropods associated

with pig carrion and human corpses in southeastern Brazil. Memórias Do Instituto Oswaldo

Cruz 95(1): 135–8

44

Carvalho LML, Linhares AX (2001) Seasonality of insects succession and pig carcass

decomposition in a natural forest area in southeastern Brazil. J Forensic Sci 46(3): 604-608

Catts EP, Goff ML (1992) Forensic entomology in criminal investigations. Ann Rev Entomol 37:

253-272

Gallagher MB, Sandhu S, Kimsey R (2010) Variation in developmental time for geo- graphically

distinct populations of the common green bottle fly: Lucilia sericata (Meigen). J Forensic Sci

55(2): 438-442

Goff ML, Lord WD (1994) Entomotoxicology: a new area for forensic investigation. Am J Forensic

Med Pathol 15: 51-57

Gram LF (1994) Fluoxetine. The New England Journal of Medicine 331(20): 1354-1361

Greenberg B, Kunich JC (2002) Entomology and the law: flies as forensic indicators. Cambridge

Universit Press, Cambridge

Guimarães JH, Prado AP, Linhares AX (1978) Three newly introduced blowfly species in

Shouthern Brazil (Diptera, Calliphoridae). Rev Bra Ent 22(1): 53-60

Hanski I (1987) Carrion fly community dynamics: patchiness, seasonality and coexistence. Ecol

Entomol 12: 257-266

Ikemoto T, Takai J. (2000) A new linearized formula for the law of total effective temperature and

the evaluation of line-fitting methods with both variables subject to error. Env Entomol 29(4):

671-682

Introna F, Campobasso CP, Goff ML (2001) Entomotoxicology. Forensic Sci Int 120: 42-47

Lü Z, Zhai X, Zhou H, Li P, Ma J, Guan L, Mo Y (2014) Effects of ketamine on the development

of forensically important blowfly Chrysomya megacephala (F.) (Diptera: Calliphoridae) and its

Forensic Relevance. J Forensic Sci, 59(4): 991-996 doi:10.1111/1556-4029.12430

R Core Team (2013) R: A language and environment for statistical computing. R Foundation for

Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0. http://www.R-project.org/

45

Richards CS, Price BW, Villet, MH (2009). 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

SAS Inc. (2006) SAS for Microsoft Windows Professional, version 9.1, Cary, NC

SAS Inc. (2009) SAS/STAT(R) 9.2 User's Guide, Second Edition, Cary, NC.

http://support.sas.com/documentation/cdl/en/statug/63033/HTML/default/viewer.htm#titlepag

e.htm Accessed 20 October 2014

Thyssen PJ and Grella MD (2011) Efeito da escopolamina sobre o desenvolvimento de Chrysomya

putoria (Diptera: Calliphoridae) e sua importância para a estimativa do intervalo pós-morte. Rev

Bras Crim 1(1): 39-42

Ullyett GC (1950) Competition for food and allied phenomena in sheepblowfly populations. Phil

Trans Royal Soc London 234: 77-174

Wells JD (1991) Chrysomya megacephala (Diptera: Calliphoridae) has reached the continental

United States: review of its biology, pest status, and spread around the world. J Med Entomol

28: 471-473

Wigglesworth VB (1972) The principles of insect physiology. Chapman and Hall, London

Wise SD (1992) Clinical studies with fluoxetine in obesity. Am J Clin Nutri 55(1): 1815-1845

Zumpt F (1965) Myiasis in man and animals in the Old World. London: Butterworths

46

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.

47

9. REFERÊNCIAS BIBLIOGRÁFICAS3

Amendt J, Krettek R, Zehner R (2004) Forensic entomology. Naturwissenschaften 91:51-65

Andrade HTA, Varela-Freire AA, Batista MJA, Medeiros JF (2005) Calliphoridae (Diptera)

coletados em cadáveres humanos no Rio Grande do Norte. Neotrop Entomol 34(5): 855–856

ANVISA (2012) Boletim de Farmacoepidemiologia. Inibidores de apetite no Brasil: reflexões

sobre seu consumo nos anos de 2009 a 2011. Sistema Nacional de Produtos Controlados

(SNGPC). Ano 2. N°1. Janeiro/ Junho de 2012. Disponível em:

http://www.anvisa.gov.br/sngpc/boletins/2012/boletim_snpgc_1_2012_modificado.pdf

Acessado em: 28 de Março de 2013

Barros-Cordeiro KB, Pujol-Luz JR (2010) Morfologia e duração do desenvolvimento pós-

embrionário de Chrysomya megacephala (Diptera: Calliphoridae) em condições de laboratório.

Papéis Avulsos de Zoologia 50(47): 709-717

Bourel B, Hédouin V, Martin-Bouyer L, Bécart A, Tournel G, Deveaux M, Gosset D (1999) Effects

of morphine in decomposing bodies on the development of Lucilia sericata (Diptera:

Calliphoridae). J of Forensic Sci 44(2): 354-358

Bourel B, Tournel G, Hédouin V, Deveaux M, Goff ML, Gosset D (2001) Morphine extraction in

necrophagous insects remains for determining ante-mortem opiate intoxication. Forensic Sci Int

120: 127-31

Byrd JH, Castner JL (2010) Forensic entomology: the utility of arthropods in legal investigations.

New York: CRC Press 2nd ed

Campobasso CP, Vella GD, Introna F (2001) Factors affecting decomposition and Diptera

colonization. Forensic Sci Int 120: 18–27

Carlini EA, Noto AR, Nappo SA, Sanchez ZM, Franco VLS, Silva LCF, Santos VE, Alves DC

(2009) Fluoxetina: indícios de isso inadequado. J Bras Psiquiatr 58(2): 97-100

3 Seguindo as normas de formatação do periódico Forensic Science International

48

Carvalho LM, Thyssen PJ, Linhares AX, Palhares FA (2000) A checklist of arthropods associated

with pig carrion and human corpses in southeastern Brazil. Memórias Do Instituto Oswaldo

Cruz 95(1): 135–8

Carvalho LMJ, Linhares AX, Trigo JR (2001) Determination of drug levels and the effect of

diazepam on the growth of necrophagous flies of forensic importance in southeastern Brazil.

Forensic Sci Int 120: 140-144

Catts EP, Goff ML (1992) Forensic entomology in criminal investigations. Annual Review of

Entomology 37: 253-272

Catts EP (1992) Problems in Estimating the Postmortem Interval. J Agric Entomol 9(4): 245–255

Chapman RF (1998) The Insects: Structure and Function. Cambridge: Cambridge University Press

4 ed

Charabidze D, Bourel B, Gosset D (2011) Larval-mass effect: Characterisation of heat emission by

necrophageous blowflies (Diptera: Calliphoridae) larval aggregates. Forensic Sci Int 211:61-66

Ferrari AC, Soares ATC, Guimarães MA, Thyssen PJ (2008) Efeito da testosterona no

desenvolvimento de Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae) Medicina

(Ribeirão Preto) 41: 30-34

França GV (2004) Medicina Legal 7 ed. Rio de Janeiro: Guanabara Koogan

Gabre RM, Adham FK, Chi H (2005) Life table of Chrysomya megacephala (Fabricius) (Diptera:

Calliphoridae). Acta Oecol 27: 179-183

Goff ML (2010) Early Postmortem Changes and Stages of Decomposition. In: Amendt, J,

Campobasso CP, Goff ML, Grassberger M (ed). Current concepts in forensic entomology.

Nehterlands: Springer

Goff ML, Lord WD (1994) Entomotoxicology: a new area for forensic investigation. Am J Forensic

Med Pathol 15: 51-57

Goff ML, Omori AI, Goodbrod JR (1989) Effect of cocaine in tissues on the development rate of

Boetcherisca peregrina (Diptera: Sarcophagidae). J Med Entomol 26: 91-93

49

Goodbrood JR, Goff ML (1990) Effects of larval population density on rates of development and

interactions between two species of Chrysomya (Diptera: Calliphoridae) in the laboratory

culture. J Med Entomol 27: 338-343

Goodman LS, Gilman A, Brunton LL, Lazo JS, Parker KL (2006) Goodman & Gilman's the

pharmacological basis of therapeutics. New York:McGraw-Hill

Gosselin M, Wille SMR, Fernandez MDMR, Di Fazio V, Samyn N, De Boeck G, Bourel B (2011)

Entomotoxicology, experimental set-up and interpretation for forensic toxicologists. Forensic

Sci Int, 208(1-3): 1–9 doi:10.1016/j.forsciint.2010.12.015

Gram LF (1994) Fluoxetine. The New England Journal of Medicine 331(20):1354-1361

Greenberg B, Kunich JC (2002) Entomology and the Law – Flies as Forensic Indicators.

Cambridge University Press, Cambridge.

Grella MD, Estrada DA, Thyssen PJ (2007) The effect of scopolamine on the development of

Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) and its importance for the post

mortem interval estimate. Entomología Mexicana 6: 870-873

Guimarães JH, Papavero N (1999) Myiasis in man and animals in the neotropical region:

bibiographic database. São Paulo: Editora Plêaide/FAPESP

Guimarães JH, Prado AP, Linhares AX (1978) Three newly introduced blowflies species in

Southern Brazil (Diptera, Calliphoridae). Rev Bras de Entomol 22:53-60

Gullan PJ, Cranston PS (2007) Os Insetos: um resumo de Entomologia. Editora Roca 3ª ed

Haddad ML, Parra JRP, Moraes RC (1999) Métodos para estimar limites térmicos inferior e

superior de insetos. Piracicaba, São Paulo: Fundação de Estudos Agrários Luiz de Queiroz

Hall RD Medicocriminal entomology. In: Catts EP, Haskell NH (1990) Entomology & Death: a

procedural guide. USA: Joyce’s Print Shop

Hanski I (1977) Biogeography and ecology of carrion flies in the Canary Islands. Ann Entomol

Fenn 43: 101-107

50

Hédouin V, Bourel B, Martin-Bouyer L, Bécart A, Tournel G, Deveaux M, Gosset D (1999)

Determination of drug levels in larvae of Lucilia sericata (Diptera: Calliphoridae) reared on

rabbit carcasses containing morphine. J Forensic Sci 44: 351-353

Hiemke C, Härtter S (2000) Pharmacokinetics of selective serotonin reuptake inhibitors. Pharmacol

Ther 85: 11–28. doi:10.1016/S0163-7258(99)00048-0

Ikemoto T, Takai J (2000) A new linearized formula for the law of total effective temperature and

the evaluation of line-fitting methods with both variables subject to error. Env Entomol 29(4):

671–682

Introna F, Campobasso CP, Goff ML (2001) Entomotoxicology, Forensic Sci Int 120: 42-47

Jirón LF, Cartín VM (1981) Insect succession in the decomposition of a mammal in Costa Rica.

Journal of the New York Entomology Society 89: 158-165

Johnson AP, Wallman JF (2014) Infrared imaging as a non-invasive tool for documenting maggot

mass temperatures. Aust J Forensic Sci 46(1): 73–79 doi:10.1080/00450618.2013.793740

Johnson AP, Wallman JF, Archer MS. (2012) Experimental and casework validation of ambient

temperature corrections in forensic entomology. J Forensic Sci. 57: 215-221

Keh B (1985) Scope and applications of forensic entomology. Ann Rev Entomol (30): 137-154

Kharbouche H, Augsburger M, Cherix D, Sporkert F, Giroud C, Wyss C, Mangin P (2008) Codeine

accumulation and elimination in larvae, pupae, and imago of the blowfly Lucilia sericata and

effects on its development. Int J Legal Med 122: 205–211 doi:10.1007/s00414-007-0217-z

Lord WD, Stevenson JR (1986) Directory of forensic entomologists. Washington DC: Reg. Prof.

Entomol.

Lü Z, Zhai X, Zhou H, Li P, Ma J, Guan L, Mo Y (2014) Effects of ketamine on the development

of forensically important blowfly Chrysomya megacephala (F.) (Diptera: Calliphoridae) and its

Forensic Relevance. J Forensic Sci, 59(4): 991-996 doi:10.1111/1556-4029.12430

51

Mégnin P (1894) La faune de cadavres. Application de l´entomologie a la medicine légale.

Encyclopédie Scientifique des Aide-Mémoire. Paris: Ed. Gauthier-Villars

Mello RS, Borja GEM, Queiroz MMC (2012) How photoperiods affect the immature development

of forensically important blowfly species Chrysomya albiceps (Calliphoridae). Parasitol Res

111: 1067-1073

Mullany C, Keller PA, Nugraha AS, Wallman JF (2014) Effects of methamphetamine and its

primary human metabolite, p-hydroxymethamphetamine, on the development of the Australian

blowfly Calliphora stygia. Forensic Sci Int 241C: 102–111. doi:10.1016/j.forsciint.2014.05.003

Nassu MP, Thyssen PJ, Linhares AX (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, Wartenberg N, Spiess R, Mall G (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

Nolte KB, Pinder RD, Lord WD (1992) Insect larvae used to detect cocaine poisoning in a

decomposed body. J Forensic Sci 37: 1179–1185

Nuorteva P (1977) Sarcosaprophagous insects as forensic indicators. In: Tedeschi CG, Eckert WG,

Tedeschi LG (ed). Forensic medicine: a study in trauma and environmental hazards.

Philadelphia, London, Toronto: W.B. Saunders Company 2: 1072-1095

Nuorteva P, Nuorteva SL (1982) The fate of mercury in sarcosaprophagous flies and in insects

eating them. Ambio 11: 34-37

Oliveira HG, Gomes G, Morlin-JR JJ, Von Zuben CJ, Linhares AX (2009) The effect of

BuscopanTM on the development of the blow fly Chrysomya megacephala (Fabricius) (Diptera:

Calliphoridae). J Forensic Sci 54(1): 202–206

52

Oliveira TC, Vasconcelos SD (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, Mello-Patiu CA, Lopes SM (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

Parry S, Linton SM, Francis PS, O´Donnell MJ, Toop T (2011) Accumulation and excretion of

morphine by Calliphora stygia, and Australian blow fly species of forensic importance J Insect

Physiol 57: 62-73

Rafael JA, Melo GAR, Carvalho CJB, Casari AS, Constantino R (2012) Insetos do Brasil:

Diversidade e Taxonomia. Ribeirão Preto: Editora Holos

Reed HB Jr (1958) A study of dog carcass communities in Tennes- see, with special reference to

the insects. Am Midl Nat 59: 213-245

Reis SF, Teixeira MA, Von Zuben FJ, Godoy WAC, Von Zuben CJ (1996) Theoretical dynamics

of experimental populations of introduced and native blowflies (Díptera: Calliphoridae). J Med

Entomol 33: 537-544

Rezende F, Alonso, MA, Souza CM, Thyssen PJ, Linhares AX (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 CS, Villet MH (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 K, Crous L, Villet, MH (2009) Models of development for blowfly sister species

Chrysomya chloropyga and Chrysomya putoria. Med Vet Entomol 23: 56-61

Schoenly K (1992) A statistical analysis of successional patterns in carrion-arthropod assemblages:

implications for forensic entomology and determination of the postmortem interval. J Forensic

Sci 37: 1489-1513

53

Sherman RA, Hall MJR, Thomas S (2000) Medical Maggots: an Ancient Remedy for some

Contemporary Afflictions. Annu Rev Entomol 45: 55-81

Slone DH, Gruner SV (2007) Thermoregulation in Larval Aggregations of Carrion-Feeding Blow

Flies (Diptera: Calliphoridae). J Med Entomol 44: 516-523

Smith KGV (1986) A manual of Forensic Entomology. Ithaca: Cornell University Press

Souza CM, Thyssen PJ, Linhares AX (2011) The effect of nandrolone decanoate on the

development of three species of Chrysomya (Diptera: Calliphoridae), flies of forensic

importance from Brazil. J Med Entomol 48(1): 11-117

Thyssen PJ (2011) Entomologia Forense. In: Marcondes CB (org.) Entomologia Médica e

Veterinária. Rio de Janeiro: Atheneu 2ªed

Triplehorn CA, Johnson NF (2004) Borror and DeLong's Introduction to the Study of Insects.

California: Thompson Brooks/Cole. Belmont 7th ed

Turner B, Howard T (1992) Metabolic heat generation in dipteran larval aggregations: a

consideration for forensic entomology. Med Vet Entomol 6: 179-181

Ullyett GC (1950) Competition for food and allied phenomena in sheepblowfly populations. Phil

Trans Royal Soc London 234: 77-174

Von Zuben CJ, Stangenhaus G, Godoy WAC (2000) Competição larval em Chrysomya

megacephala (F.) (Diptera: Calliphoridae): efeitos de diferentes níveis de agregação larval sobre

estimativas de peso, fecundidade e investimento reprodutivo. Rev Bras Biol 60: 195-199

Wagner TL, Wu H, Sharpe PJH, Schoolfield RM, Coulson RN (1984) Modeling insect

development rates: a literature review and application of a biophysical model. Ann Entomol Soc

Am 77: 208–225

Wells JD, B Greenberg (1992) Rates of predation by Chrysomya rufifacies (Macquart) on

Cochliomyia macellaria (Fabr.) (Diptera: Calliphoridae) in the laboratory: effect of predator and

prey development. Pan. Pac. Entomol 68: 12-14

54

Wells JD, Kurahashi H (1994) Chrysomya megacephala (Fabricious) (Diptera: Calliphoridae)

development: rate, variation and implications for forensic entomology. Japanese J Sanitary Zool

45(4): 303–309

Wells JD (1991) Chrysomya megacephala (Diptera: Calliphoridae) has reached the continental

United States: review of its biology, pest status and spread around the world. J. Med. Entomol.

28: 471–473

Wigglesworth VB (1972) The principles of insect physiology. Chapman and Hall, London

Wilcox JA (1987) Abuse of fluoxetine by a patient with anorexia nervosa. Am J Psychiatry

144(8):1100

Zhang ZQ (2013) Phylum Arthropoda. In: Zhang ZQ (ed.) Animal Biodiversity: An outline of

higher-level classification and survey of taxonomic richness (Addenda 2013). Zootaxa 3703:1–

82

Zumpt F (1965) Myiasis in man and animals in the Old World. London: Butterworths

55

10. ANEXO