Biología Reproductiva de Hermetia Illucens

Biologa reproductiva y caracterizacin morfolgica
de los estadios larvarios de Hermetia illucens

(L., 1758) (Diptera: Stratiomyidae). Bases
para su produccin masiva en Europa
Flavia Paola Gobbi

Biologa reproductiva y caracterizacin morfolgica de los
estadios larvarios de Hermetia illucens (L., 1758) (Diptera:
Stratiomyidae). Bases para su produccin masiva en Europa.
Flavia Paola Gobbi
Centro Iberoamericano de la Biodiversidad
Instituto Universitario de Investigacin, Universidad de Alicante.
Programa de Doctorado: Biodiversidad Gestin y Conservacin de las Especies
Y sus Hbitat
Diciembre de 2012
iii
Biologa reproductiva y caracterizacin morfolgica de los
estadios larvarios de Hermetia illucens (L., 1758) (Diptera:
Stratiomyidae). Bases para su produccin masiva en Europa.
Tesis Doctoral presentada por la Licenciada en Biologa Flavia Paola Gobbi para optar
al ttulo de Doctor en Biologa por la Universidad de Alicante
Directores:
Dr. Santos Rojo Velasco Dra. Ana Isabel Martnez Snchez

Instituto CIBIO/Dpto. CARN Instituto CIBIO/Dpto. CARN
Universidad de Alicante Universidad de Alicante
Alicante, 2012
v
A Guillermo y
a mis padres,
Beatrz y Delmar
vii
AGRADECIMENTOS
Esta tesis no habra sido posible sin el apoyo de muchas personas, por lo
que me gustara expresar mi agradecimiento a los siguientes colegas, amigos y
familiares.
En primer lugar agradezco a mis directores, el Dr. Santos Rojo y la Dra.

Ana Isabel Martnez-Snchez, quienes me facilitaron da a da todos los
conocimientos y las herramientas necesarias para desarrollar las investigaciones
que llevaron a la presente tesis doctoral. Tambin agradecerles la confianza que
depositaron en mi para la realizacin de todas las tareas de investigacin que se
llevaron a cabo durante este proyecto, siendo muchas de ellas de gran
envergadura. La amistad y el aliento proporcionados por cada uno de ellos han
sido indispensables para el trabajo del da a da, as como tambin la paciencia
que necesitaron muchas veces. Gracias al aprendizaje con cada uno de ellos, me
siento capacitada y formada como investigadora para afrontar el camino de mi
siguiente etapa.
Me gustara dar las gracias a la empresa Flysoil S. A. ya que han

contribuido a financiar parte de la presente tesis, as como tambin el haber
compartido algunos de sus conocimientos que facilitaron la comprensin de
algunos aspectos biolgicos de Hermetia illucens.
Al Ministerio de Ciencia e Innovacin por otorgarme la beca

Subprograma Torres Quevedo 2010 y 2011, importante financiamiento para la
realizacin de esta tesis doctoral.
Tambin quisiera agradecer a la empresa Agriprotein S. A. por permitirme

compartir mis conocimientos, otorgndole peso a toda la investigacin realizada.
ix
Me gustara dar las gracias a todos los miembros del grupo de
investigacin Bionoma, Sistemtica e Investigacin Aplicada de Insectos
Dpteros e Himenpteros de la Universidad de Alicante (Celeste, Tania,
Esperanza, Elena), porque sin su apoyo no habra sido lo mismo; en especial a
Yelitza, Berta y Pilar por todos los momentos distendidos que hemos compartido,
as como la amistad que hemos ido formando da a da.
Tambin tengo que agradecer a los directivos, administrativos y al

personal tcnico del CIBIO, ya que su apoyo ha facilitado la realizacin de este
proyecto. En especial a Antonio, David y Yolanda por solucionarme numerosos
problemas de papeleo; a Chema por ayudarme a resolver muchos problemas
tcnicos de informtica y por ltimo a Carmen y Jess que han sido un pieza
clave, ya que han aportado ideas fundamentales para el diseo experimental de la
tesis.
Especial agradecimiento al personal de los Servicios Tcnicos de

Investigacin de la Universidad de Alicante; en especial al Dr. Pablo Candela
Antn de la unidad de espectrometra de masas, a Vernica Lpez Belmonte de
la unidad de microscopa y a Sara Alcaiz Lucas y Jos Luis Carbonell Garrigs
del rea de infraestructuras de apoyo, por la paciencia y ayuda que han
proporcionado para el ptimo desarrollo de esta tesis doctoral.
A todos mis amigos del CIBIO, que gracias a su compaa y a todos esos
buenos momentos, mi trayectoria en el departamento durante la realizacin de
esta tesis ha resultado ms llevadera; no hubiera sido lo mismo sin ellos.
Por ltimo, me gustara expresar mi ms profundo agradecimiento a mi

familia por su inquebrantable amor y apoyo. Esta tesis no habra sido posible sin
ellos, ya que son una base fundamental en mi vida. A mis padres, que me han
enseado los valores importantes de la vida, y que me han enseado que sin
lucha no hay recompensa, no hay palabras que puedan expresar mi gratitud hacia
ellos. A Guillermo, mi compaero de vida, sin el nada de esto hubiera podido
x
hacerse realidad, siempre ha permanecido a mi lado apoyndome
incondicionalmente sin esperar nada a cambio; l hace que sea mejor persona.
xi
xii
NDICE
RESUMEN........1
ABSTRACT......6
INTRODUCCIN....7
1. Antecedentes y generalidades.9
1.1. Parmetros biolgicos de Hermetia illucens......13
1.2. Importancia econmica de Hermetia illucens....16
1.3. Hermetia illucens como agente implicado en el clculo del intervalo

postmortem (IPM) en entomologa forense...18
2. Objetivos y estructura de la tesis..........19
3. Bibliografa...............28
CAPTULO I: Estudio de la morfologa larvaria y anlisis preliminar de la

variacin de los hidrocarburos cuticulares durante el desarrollo preimaginal
de Hermetia illucens (L.) (Diptera, Stratiomyidae)..........................................29
1. Introduccin..........32
2. Material y Mtodos...........35
2. 1. Caracterizacin morfolgica de las larvas.....36
2. 2. Cuantificacin de hidrocarburos cuticulares..52
2. 3. Anlisis estadstico....53
3. Resultados.............53
3. 1. Anlisis morfolgico..53
xiii
3. 2. Anlisis de hidrocarburos cuticulares....60
4. Discusin..............65
5. Bibliografa...............69
CAPTULO II: Growing curves of the Black Soldier Fly, Hermetia illucens
(Diptera: Stratiomyidae) in two different larvae media. ...77
1. Introduction...............80
2. Methodology.............81
3. Results...............83
4. Discussion.................92
5. Bibliography.................96
CAPTULO III: The effects of larval diet on adult life-history traits of the
Black Soldier Fly, Hermetia illucens (L.) (Diptera, Stratiomyidae). .......101
1. Introduction.............104
2. Methodology...........105
2.1. Experimental design.............106
2.2. Statistical analysis.............108
3. Results.............109
3.1. Mortality, duration of stages and sex-ratio...........109
3.2. Adult size and ovarian development.......111
4. Discussion...........118
5. Bibliography...............121
xiv
CAPTULO IV: Mass rearing of Hermetia illucens (Diptera:
Stratiomyidae): identifying bottlenecks in egg production. .........127
1. Introduction.............130
2. Methodology...........132
2.1. Adult density experime.............133
2.2. Mass rearing experiment...........134
2.3. Data analysis and statistic.............134
3. Results.............137
3.1. Effect of density on the production of eggs......137
3.2. Experiment mass rearing with different protocols....141
4. Discussion...................145
5. Bibliography...................148
CONCLUSIONES........153
xv
xvi
RESUMEN
Hermetia illucens (Linnaeus, 1758) es un dptero estratiomido (Diptera,

Stratiomyidae) vulgarmente denominado mosca soldado negra (Black Soldier
Fly, BSF en ingls) de origen posiblemente neotropical pero actualmente est
presente en zonas clidas de todo el mundo, debido a su transporte accidental o a
su introduccin deliberada con diferentes usos. La especie es susceptible de ser
criada a escala masiva y los estadios larvarios pueden alimentarse de multitud de
restos orgnicos de muy diverso origen. Es por ello que esta especie presenta un
gran inters desde un punto de vista aplicado ya que por su versatilidad puede ser
utilizada tanto para la transformacin de residuos/subproductos orgnicos en
biomasa til para la alimentacin animal o la obtencin de biomolculas, como
bioindicador forense por su papel en investigaciones forenses y su uso para el
clculo del intervalo postmortem. Por todos estos motivos se necesita una
informacin profunda sobre la morfologa, biologa y ecologa de H. illucens y
en particular sobre los parmetros biolgicos asociados a su cra artificial y
produccin masiva. Con el fin de obtener y analizar estos conocimientos se
propuso la realizacin de la presente tesis doctoral, incidiendo especialmente en
la situacin de su cra en Europa. Los principales parmetros estudiados se
abordaron en diferentes captulos resumidos a continuacin.
Se analiz la morfologa de los diferentes estadios larvarios y fases

preimaginales, prestando especial atencin a la quetotaxia, el tamao de la
capsula ceflica y la caracterizacin morfolgica de los espirculos anteriores y
posteriores. No se observaron diferencias sustanciales en la quetotaxia ni en lo
relativo a los espirculos anteriores de las larvas de diferentes edades; sin
embargo, en el tamao de la capsula ceflica y la morfologa de los espirculos
posteriores ocurri lo contrario, detectndose caractersticas diagnsticas vlidas
-1-
para los diferentes estadios larvarios. Tambin, se presentan los resultados de la
caracterizacin bioqumica de los hidrocarburos presentes en la cutcula del
exoesqueleto de los diferentes estadios larvales. En este sentido, pudo
comprobarse que a medida que aumenta la edad de las larvas, aumenta de manera
progresiva la abundancia de diferentes compuestos hidrocarbonados. Este hecho
puede ser utilizado en diversas vertientes del mbito aplicado como por ejemplo
la estimacin de la edad en el clculo del intervalo postmortem o su aplicacin
como factor de control de calidad en la produccin masiva de H. illucens con
diversos fines industriales.
A continuacin, se determinaron los requerimientos trmicos o suma

trmica segn el modelo de grados-da de la especie para su posterior empleo en
investigacin aplicada. Esta tcnica permite relacionar cada fase de desarrollo
con una acumulacin de unidades trmicas, sobre una temperatura umbral (la
constante trmica). Se estudi el efecto de la temperatura y el desarrollo
preimaginal de H. illucens sobre diferentes medios de alimentacin. Entre los
parmetros analizados se encuentran el tiempo de desarrollo y el efecto en el
crecimiento larvario (longitud y peso), a tres temperaturas constantes (25, 30 y
35 C). Con los datos de desarrollo se calcul la temperatura mnima de
desarrollo (T0) y se elabor un diagrama isomorfo para cada medio de desarrollo
(carne de cerdo y pienso de gallina ponedora).
Tambin se analiz el efecto de tres medios de desarrollo larvario (pienso

de gallina ponedora, harina crnica multiespecie y harina crnica mezclada con
pienso de gallina ponedora) en diferentes parmetros biolgicos de los imagos
obtenidos como el tamao alar (analizado mediante morfometra geomtrica) y el
desarrollo ovrico de las hembras de H. illucens. Se encontraron diferencias
significativas en el tamao alar de los imagos obtenidos en su fecundidad,
mortalidad, y otros parmetros estudiados,
Por ltimo, con objeto de identificar y resolver los cuellos de botella

relacionados con la produccin masiva de huevos de H. illucens en condiciones
de cra artificial, se determinaron los principales factores abiticos y biticos
-2-
relacionados con el desarrollo y maduracin de los imagos estableciendo los
lmites de la produccin de huevos. Los principales resultados indican que tanto
la luz solar como la densidad de adultos y el tamao de la caja de cra, tienen una
influencia significativa sobre el desarrollo del ciclo biolgico de la especie.
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-4-
ABSTRACT
Hermetia illucens (Linnaeus, 1758) is a dipterous stratiomido (Diptera,

Stratiomyidae) commonly called "black soldier fly" (BSF), with neotropical
origin that currently occurs in warm worldwide areas because of accidental
transportation or intentional introduction of different uses. The species is capable
of being raised on a massive scale, the larval stages have a higher resistance
feeding a large amount of organic matter of diverse origin. The versatility of this
species can be used for processing of waste/biomass byproducts into useful
organic feed or obtaining biomolecules and to use in forensic investigations for
calculating the postmortem interval. For all these reasons it takes a deep
information on the morphology, biology and ecology of H. illucens particularly
on biological parameters associated with its artificial breeding and mass
production. In order to obtain and analyze this knowledge is proposed to hold
this thesis, with special emphasis on the status of their breeding in Europe. The
main parameters studied were addressed in different chapters summarized below.
Were analyzed the morphology of the different larval stages and phases
preimaginal, paying particular attention to the chaetotaxy, the size of the head
capsule and morphological characterization of the anterior and posterior
spiracles. No substantial differences were observed in the chaetotaxy and in the
previous spiracles larvae of different ages, however, the head capsule size and
morphology of the posterior spiracles showed different characteristics with
respect to the age of the larvae, valid for features diagnostic of different larval
stages. We present the results of the biochemical characterization of the
hydrocarbons in the cuticle of different larval stages. In this regard, it was found
that with increasing age of the larvae, progressively increases the abundance of
different hydrocarbon compounds. This fact can be used in various areas of
-5-
applied field such as age estimation in the postmortem interval calculation factor
or its application as quality control in mass production of H. illucens with various
industrial purposes.
The following requirements were determined thermal or heat summation

by degree-day model of the species for later use in applied research. This
technique relates each development phase with an accumulation of heat units
above a threshold temperature (constant temperature). Was studied the effect of
temperature on different media of larval development of H. illucens. Was
analyzed the development time and the effect on larval growth (length and
weight) on three constant temperatures (25, 30 and 35 C). With the
development data was calculated minimum temperature development (T0) and
developed a diagram isomorphic to each development environment (pork meat
and hen feed).
We also analyzed the effect of the media in the larval development (hen
feed, meat meal+hen feed mixed and meat meal alone) on different biological
parameters as adults wing size (analyzed by geometric morphometrics) and
ovarian development H. illucens females. Significant differences in the wing
size, in fertility, mortality, and other parameters studied, was obtained.
Finally, in order to identify and resolve bottlenecks related to the mass

production of eggs of H. illucens in artificial rearing conditions, were determined
the abiotic and biotic factors related to the development and maturation of adults.
The main results show that both, sunlight and adult density and size of the
breeding box, have a significant influence on the development of the life cycle of
the species.
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INTRODUCCIN GENERAL
-8-
Generalidades de Hermetia illucens
1. Antecedentes y generalidades
El orden Diptera constituye uno de los principales grupos de insectos,

presentando aproximadamente 100 familias descritas y ms de 85.000 especies
conocidas. Una gran parte de estos insectos presentan gran importancia
econmica, bien sea por su importante papel en la descomposicin de la materia
orgnica, por actuar como fauna til en el control de plagas o por su papel como
agentes polinizadores tanto en agrosistemas como hbitats naturales. Por otro
lado, algunos grupos, especialmente aquellos con hbitos hematfagos, son
importantes vectores de diversos agentes infecciosos en el mbito mdico-
veterinario (Borror et al., 1976).
Los Brachycera son el grupo ms diversificado de dpteros, y a l

pertenece la familia Stratiomyidae, con alrededor de 2.600 especies descritas
integradas en aproximadamente 400 gneros (Woodley, 2001). La familia
Stratiomyidae se encuentra presente en todas las regiones biogeogrficas del
planeta, estando sus larvas en diversos tipos de hbitats, aunque preferentemente
en zonas hmedas o saturadas de agua, en el medio edfico, bajo cortezas, y en
materia orgnica en descomposicin de diversos orgenes. Los imagos presentan
una llamativa diversidad morfolgica superior al resto de familia de dpteros, y
normalmente se localizan sobre la vegetacin cercanos a los lugares de desarrollo
larvario (Borror et al., 1976; Woodley, 2001). Las larvas presentan significativas
caractersticas diagnsticas, con el tegumento endurecido por depsitos
calcreos, el cuerpo aplanado dorso-ventralmente, y en ocasiones un sifn corto
al final del cuerpo (James, 1981).
Hermetia illucens (Linnaeus, 1758) (Figura 1) conocida como mosca

soldado (= Black Soldier Fly) es un Stratiomyidae, posiblemente originario del
Nuevo Mundo (Kovac & Rozkosny, 1995) pero que a causa de la actividad
-9-
Introduccin general
humana se ha distribuido por todas las regiones tropicales hmedas y

subtropicales del planeta (James, 1935). Sin embargo, pueden tolerar
temperaturas extremas (Callan, 1973), aunque no durante el momento de la
ovoposicin (Drees & Jakman, 1998). En Europa, se registr por primera vez en
Malta en 1926, y desde entonces, se ha citado en amplias zonas de la regin
Mediterrnea, Albania, Croacia, Francia, Italia, el sur de Suiza, Portugal y
Espaa (Martnez-Sanchz et al., 2011). En la pennsula Ibrica, H. illucens se
registr por primera vez en 1954 en Espaa, y en Portugal en 1995 (Martnez-
Snchez et al., 2011).
Figura 1. Adulto de Hermetia illucens (de www.CritterZone.com).
Los adultos, probablemente presentan una dieta florcola en condiciones

naturales, pero en cautividad pueden sobrevivir varias semanas sin alimento
(Tomberlin et al., 2002). Por el contrario, las larvas pueden desarrollarse en una
amplia diversidad de materia orgnica, desde estircol y carne en
descomposicin, hasta frutos y vegetales; por otra parte, en ocasiones pueden
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causar miasis accidental en el ser humano (James, 1947; Caldern-Arguedas et

al., 2005).
Los imagos son muy variados en forma y coloracin, presentando un

mimetismo con ciertos grupos de himenpteros, que en principio les confiere sus
ventajas ante ciertos depredadores. Otra particularidad son sus conspicuos ojos
dicpticos. La hembra suele presentar un tamao superior al macho, aunque no
existe un evidente dimorfismo sexual. El aparato genital fue descrito por primera
vez por Rozkosny (1983) (Figura 2). La genitalia masculina es relativamente
corta y presenta dos pares de lbulos posteriores laterales, un par de cercos y un
par de gonostilos muy reducidos. El complejo edeagal es muy delgado y se
encuentra dilatado en su parte basal. La terminalia femenina se compone de un
par cercos largos formados por dos segmentos; posee una larga placa subgenital
en su parte distal de forma puntiaguda y una furca genital subtriangular (stner
et al., 2003). La estructura genital representa el nico carcter de dimorfismo
sexual de esta especie (Figura 3).
Los imagos presentan una pigmentacin predominantemente oscura, con

alas de color marrn o negro. El tamao de las antenas es al menos dos veces la
longitud de la cabeza y estn constituidas por ocho artejos irregulares; el ltimo
flagelmero, presenta la arista. Las patas son principalmente negras, aunque en la
zona basal de todos los tarsos se observa una pigmentacin blanca. El abdomen
consta de cinco segmentos visibles de color negro, pero en la parte posterior del
margen de los terguitos 1 y 2 se encuentran un par de manchas (tambin
llamados espejos=mosca de espejuelos) translcidos, blancos y oblongos con
funcin desconocida (stner et al., 2003) (Figura 1).
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Introduccin general
Figura 2. 53-55: Genitalia masculina de un Stratiomyidae; 53: vista dorsal; 54: vista lateral; 55
vista ventral. 56-58: Terminalia femenina de un Stratiomyidae; 56: vista dorsal; 57: furca
genital; 58: vista ventral. (aed: complejo edeagal, cerc: cerco, ep: epandrium, epipr: epiprocto,
gcx: gonocoxito, gcxap: apodema gonocoxal, gst: gonostylus, S: esternito, synst: synsternito, T:
terguito) (Rozokosny 1983).
A B
Figura 3. Vista ventral del final del abdomen en un macho (A) y hembra de Hermetia illucens.
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1.1. Parmetros biolgicos de Hermetia illucens
Aproximadamente cinco das despus de la emergencia del adulto puede

ocurrir la cpula. Tingle y colaboradores (1975) describieron la conducta de
apareamiento de esta especie indicando que las hembras que se encuentran en
reposo atraen a los machos en vuelo, de manera que estos descienden para la
cpula. Sin embargo, Copello (1926) seal previamente que el apareamiento se
produca durante el vuelo y no en reposo. Tomberlin y Sheppard (2001)
proporcionaron una nueva descripcin del apareamiento de esta especie. Segn
estos autores, el macho intercepta a la hembra en el aire descendiendo luego en
copula, en determinados lugares que son defendidos contra otros machos
(sistema de lekking).
Tras el apareamiento las hembras depositan alrededor de 600 huevos en

grietas o hendiduras cerca de materia orgnica en descomposicin (Sheppard,
1983). Cada huevo con forma de valo mide aproximadamente 1 mm de
longitud, su coloracin vara de blanco a amarillo plido o crema, emergiendo
larvas de primer estadio en aproximadamente cuatro das a 24 C (Booth &
Sheppard, 1984). Las larvas son de crecimiento rpido y se caracterizan por seis
estadios larvales (L1, L2, L3, L4, L5 y prepupa). Las larvas pueden llegar a
medir hasta 3 cm de longitud, son de un color opaco y blanquecino y presentan
una caracterstica quetotaxia tanto en su parte ventral como dorsal (Hall &
Gerhardt, 2002). Al finalizar su crecimiento, las larvas abandonan el medio de
desarrollo buscando un sitio seco y protegido, a este estadio se lo denomina
prepupa (sexto estadio larval). Esta etapa se caracteriza por el endurecimiento y
oscurecimiento de la cutcula, as como por su gran movilidad y tras unos das se
transforma en pupa caracterizada por la falta de movimiento activo (Hall &
Gerhardt, 2002).
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Introduccin general
Los adultos emergen aproximadamente despus de dos semanas tras la

formacin de la prepupa (Tomberlin et al., 2002). Los adultos no necesitan
alimentarse por lo que dependen de las reservas acumuladas durante la fase
larvaria (Newton et al., 2005). La duracin de cada etapa del ciclo de vida de H.
illucens est influenciado por diversos factores abiticos y biticos, que pueden
alterar de forma significativa el desarrollo de las etapas preimaginales de esta
especie (Tomberlin & Sheppard, 2002).
En Hermetia illucens al igual que en la mayor parte de los insectos, la

temperatura afecta directamente sobre el crecimiento y desarrollo de las etapas
preimaginales independientemente de la disponibilidad de alimento (Gullan &
Cranston, 2000). El desarrollo de un insecto se puede describir mediante una
curva de rendimiento trmico, donde desde una temperatura mnima su desarrollo
aumenta hasta una temperatura ptima, disminuyendo rpidamente a una
temperatura mxima (Deutsch et al., 2008). Las temperaturas mnima y mxima
se denominan umbrales de desarrollo y cuando los insectos se enfrentan a
entornos ambientales ms all de sus umbrales de desarrollo, ste se relentiza o
detiene. Las temperaturas ptimas para el ciclo biolgico de H. illucens se sitan
en el rango 24 a 29,3 C (Furman et al., 1959; Tingle et al., 1975; Bradley &
Sheppard, 1983; Booth & Sheppard, 1984; Sheppard & Newton, 2000).
Al igual que la temperatura, la humedad ambiental puede tener

importantes efectos fisiolgicos afectando al desarrollo, longevidad y la
oviposicin de H. illucens (Gullan & Cranston, 2000). La cutcula del
exosesqueleto est formada por un capa lipdica superficial impermeable al agua
(Wigglesworth, 1944). La tasa de transpiracin a travs de la cutcula en especies
adaptadas a climas hmedos tiende a ser superior a las de ambientes ms secos
(Wigglesworth, 1984). Por ello es importante conocer los mecanismos
conductuales empleados por los insectos con este fin. En particular, se han
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descrito diversas estrategias durante la ovoposicin, tales como la agrupacin de

huevos en masa y la seleccin del sitio de oviposicin, por ejemplo en la parte
inferior de una hoja hmeda cerca de la fuente de alimento. Las hembras de H.
illucens suelen ovopositar en grietas secas cerca de un recurso hmedo (Booth &
Sheppard, 1984), de manera que larvas recin eclosionadas pueden rpida y
fcilmente abrirse camino hacia el recurso antes de la desecacin. Adems, las
larvas tambin se encuentran amenazadas por la prdida de agua corporal en un
ambiente terrestre (Gullan & Cranston, 2000). Estudios de laboratorio con H.
illucens determinaron que el rango ptimo para el desarrollo de la especie es 50
a 99% de humedad relativa del aire (Furman et al., 1959; Tingle et al., 1975;
Bradley & Sheppard, 1983; Booth & Sheppard, 1984).
Por otro lado, algunos estudios indican que determinadas caractersticas

lumnicas estimulan el apareamiento de los adultos, en particular se ha propuesto
que los ojos de H. illucens presentan caractersticas particulares nicas de
fotorrecepcin (Tomberlin & Sheppard, 2002; Zhang et al., 2010).
Otros factores como la calidad y cantidad de alimento as como tambin la

densidad poblacional son de vital importancia en el desarrollo de esta especie
(Sheppard et al., 2002; Tomberlin & Sheppard, 2002). Segn Liu y
colaboradores (2008) la cantidad de alimento diario que requieren las larvas para
su adecuado crecimiento depende de su contenido nutricional (Sheppard et al.,
2002). En condiciones ideales, las larvas tardan dos semanas en alcanzar el
estado de prepupa, pero si hay limitaciones de alimento este perodo se puede
extender hasta cuatro meses (Furman et al., 1959). Esta habilidad para extender
el estado larval en respuesta a la disponibilidad de alimento aumenta las
posibilidades de supervivencia a largo plazo en condiciones naturales (Sheppard
- 15 -
Introduccin general
et al., 1994) permitiendo su adaptacin a diferentes tipos de hbitats y medios de

desarrollo.
Para la cra en cautividad de Hermetia illucens, Tingle y colaboradores

(1975) sealaron que el apareamiento y la ovoposicin solo se lograba en cajas
de colonia de 3 x 6,1 x 1,8 m y 0,76 x 1,14 x 1,37 m. Sin embargo, Sheppard y
colaboradores (2002) obtuvieron resultados similares en cajas de colonia de 2 x 2
x 4 m. Estos resultados podran estar relacionados con la necesidad de espacio de
los imagos para el adecuado desarrollo del comportamiento tipo lekking
(Tomberlin et al., 2002).
1.2. Importancia econmica de Hermetia illucens
Uno de los principales retos del siglo XXI es la bsqueda de una solucin
en la gestin sostenible de los residuos orgnicos, especialmente en ambientes
urbanos y tambin en el mbito agroalimentario. Como se ha mencionado
anteriormente, las larvas de H. illucens, pueden alimentarse en diversos tipos de
residuos orgnicos. Esta versatilidad puede ser empleada para obtener excelentes
resultados en la eliminacin de residuos orgnicos (Lard, 1989; Newton et al.,
2005a; St-Hilaire et al., 2007; Hem et al., 2008).
La gestin de restos orgnicos mediante insectos trasforma estos en

biomasa reutilizable de diversas maneras, siendo una de las que presenta
mejores perspectiva como alimento animal. Las larvas de H. illucens pueden ser
utilizadas como fuente de alimento para aves de corral (Sheppard et al., 2002).
Su alta concentracin proteica y otros nutrientes como: cidos grasos, pigmentos,
vitaminas y/o minerales, permiten su inclusin en las dietas en avicultura,
ganadera y acuicultura. Sheppard y colaboradores (2002) evaluaron el uso de
larvas o harinas de larvas de H. illucens en ensayos con pollos, cerdos y peces,
- 16 -
demostrando su utilidad como fuente de protena cruda y lpidos altamente

deseables con cadenas medias de cidos grasos monosaturados. Estudios con
harina de larvas de H. illucens para la alimentacin de peces ha revelado
resultados prometedores en lo que respecta a la sustitucin de la harina de
pescado (Hale, 1973; Newton et al., 1977; Bondari & Sheppard, 1987). Newton y
colaboradores (2005b) sustituyeron el 50% de la harina de pescado comercial con
harina de larvas de esta especie sin efectos negativos sobre el crecimiento de
alevines de la especie Ictalarus punctatus (bagre de canal o pez de gato
americano).
Otro interesante subproducto derivado de la utilizacin de larvas de H.

illucens procede de su exoesqueleto; la cutcula de los insectos se compone de
quitina adems de lpidos y otros compuestos. La quitina es de inters comercial
(quitosano) debido a su alto porcentaje de nitrgeno (6,9%). Sin embargo, la
viabilidad econmica de la extraccin de quitina de prepupas de H. illucens
todava debe ser evaluada.
Una ventaja adicional de H. illucens es su capacidad para repeler la

oviposicin de Musca domestica (Bradley & Sheppard, 1984), un transmisor
mecnico de enfermedades especialmente importante en los pases en desarrollo,
donde la falta de saneamiento y de agua corriente implican fuentes potenciales
de agentes patgenos (Graczyk et al., 2001). En este sentido el empleo de larvas
de mosca soldado en la conversin de bio-estircol disminuy los niveles de
Escherichia coli (Erickson et al., 2004; Liu et al., 2008); esta capacidad sin
embargo, est muy influenciada por la temperatura obteniendo una tasa de
reduccin ptima entre 27 C y 31 C (Liu et al., 2008). Los autores observaron
que aunque la presencia de larvas disminuye los recuentos de bacterias, no las
elimina por completo (Liu et al., 2008).
- 17 -
Introduccin general
Por otro lado, la actividad larvaria en conjuncin con la actividad

bacteriana, no slo reducen la masa seca, sino tambin otros componentes tales
como el nitrgeno o fsforo. Experimentos con estircol de vaca mostraron una
reduccin de 43% de nitrgeno y 67% de fsforo (Myers et al., 2008). La
combinacin de la capacidad de tratamiento de residuos junto con la generacin
de un producto de valor econmico hace que esta especie sea una herramienta
muy prometedora para la gestin de residuos orgnicos.
1.3. Hermetia illucens como agente implicado en el clculo del intervalo

postmortem (IPM) en entomologa forense
Los cuerpos de los animales en descomposicin son fuente de alimento

temporal para diversos organismos tales como hongos, bacterias, artrpodos e
incluso vertebrados (Smith, 1986). En este microhbitat, los artrpodos son los
principales colonizadores siendo los insectos sarcosaprfagos la fauna
predominante (Nuorteva, 1977). As, la informacin acerca de los insectos, en
combinacin con otros procedimientos forenses, generan datos que pueden ser
tiles en las investigaciones forenses cuyo objetivo principal es determinar el
intervalo postmortem (IPM), es decir, el tiempo transcurrido desde la muerte
hasta el descubrimiento del cuerpo, y las inferencias sobre la ubicacin, el modo
o la causa de la muerte (Haskell & Catts, 1990; Catts & Goff, 1992). La
importancia de utilizar los insectos en investigaciones criminales reside en el
hecho de que a menudo son los primeros en llegar al cadver despus de la
muerte y pueden permanecer en todas las etapas de descomposicin (Caravalho
& Ribeiro, 2000). Entre los muchos factores extrnsecos que influyen en el
proceso de descomposicin estn las condiciones ambientales tales como la
temperatura, la humedad, la disponibilidad de oxgeno, la ubicacin y el estado
- 18 -
del cuerpo, si estn intactos o mutilados, etc (Ubelaker, 1997). Tambin existen
factores intrnsecos como sustancias qumicas (Guimares et al., 1978).
Hermetia illucens puede clasificarse como necrfaga oportunista o

secundaria (Haskell & Catts, 1990; Lord et al., 1994). No obstante, se ha
demostrado que esta especie puede resultar muy til para el clculo del IPM
(Lord et al., 1994; Oliveira-Costa, 2003; Pujol-Luz et al., 2008), sobre todo para
muertes de ms de 15 das. Son varios los trabajos que recientemente, han
demostrado la importancia forense de H. illucens en Europa (Turchetto et al.,
2001; Martnez-Snchez et al., 2011).
2. Objetivos y estructura de la tesis
El objetivo general de esta tesis doctoral es el anlisis de los principales

parmetros biolgicos involucrados con la cra y produccin masiva de H.
illucens en condiciones controladas as como su uso como indicador forense. El
conocimiento de su biologa reproductiva as como la caracterizacin
morfolgica de sus estadios preimaginales facilitarn el empleo de H. illucens en
diversos mbitos de la investigacin aplicada, como su uso en alimentacin
animal o en el clculo del intervalo posmortem (IPM) en entomologa forense.
Para llevar a cabo este objetivo general se plantearon diversos objetivos

especficos que se desarrollaron en los siguientes captulos de esta tesis:
Captulo I: Caracterizacin morfolgica y variacin en los

hidrocarburos cuticulares de H. illucens, durante su desarrollo
preimaginal. Mediante tcnicas de microscopa ptica convencional y
microscopa electrnica, se analizaron los cambios morfolgicos
- 19 -
Introduccin general
acontecidos durante el desarrollo larvario. Tambin se realizo el anlisis

de la composicin qumica del exoesqueleto cuticular de los estadios
larvarios en funcin de la edad de desarrollo y su utilidad en investigacin
aplicada.
Captulo II: Clculo de la suma trmica (growing-degree days = suma

de grados-da) durante el desarrollo preimaginal de H. illucens.
Estudio de la duracin de las etapas preimaginales a diferentes
temperaturas y en medios de desarrollo larvario distintos.
Captulo III: Influencia del medio de desarrollo larvario en la eficacia

biolgica de H. illucens. Evaluacin del efecto de la alimentacin y
desarrollo preimaginal en la mortalidad, fecundidad, tamao y otros
parmetros biolgicos de los imagos.
Captulo IV: Identificacin de cuellos de botella en la produccin

masiva de huevos de H. illucens en condiciones controladas.
Determinacin de los factores abiticos y biticos clave que afectan al
desarrollo imaginal, determinando los lmites de la produccin masiva de
huevos de la especie.
- 20 -
3. BIBLIOGRAFA
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entomology. London, United Kingdom: Blackwell Science.
HALE, O. M. 1973. Dried Hermetia illucens larvae (Diptera: Stratiomyidae) as a

feed additive for poultry. J. Georgia Entomol. Soc. 8:16-20.
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palm kernel meal for aquaculture: Experiences from the forest region
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Guide, Joyces Print Shop, Clemson, SC. pp 52-97.
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Stratiomyidae). Bulletin of the Brooklyn Entomological Society. 30:165-
170.
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JAMES, M. T. 1981. Stratiomyidae. Chapter 36. In MCALPINE, J. F.; B. V.

PETERSON, B. V.; SHEWELL, G. E.; TESKEY, H. J.; VOCKEROTH,
J. R. & WOOD, D. M. Manual of Nearctic Diptera. Ottawa: Research
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KOVAC, D. & ROZKOSNY, R. 1995. Stratiomyidae (Insecta: Diptera) of

Temengor Forest Reserve, Hulu Perak, Malaysia. Malayan Nature
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LARD, G. 1989. Investigation on some factors affecting larval growth in a

coffee-pulp bed. Biological Wastes. 30:11-19.
LINNAEUS. C. 1758. Systema naturae per regna tria naturae. Ed. 10. Vol. 1, l
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LIU, Q. L.; TOMBERLIN, J. K.; BRADY, J. A.; SANFORD, M. R. & YU, Z. N.

2008. Black Soldier Fly (Diptera: Stratiomyidae) Larvae Reduce
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black soldier fly Hermetia illucens (Diptera, Stratiomyidae) as a potential
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Development of Black Soldier Fly (Diptera: Stratiomyidae) Larvae Fed
Dairy Manure. Environ. Entomol. 37(1):11-15.
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Vestgios. So Paulo, Millennium, 180 p.
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Introduccin general
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Captulo I
Caracterizacin larvaria de Hermetia illucens
Estudio de la morfologa larvaria y anlisis preliminar

de la variacin de los hidrocarburos cuticulares,
durante el desarrollo preimaginal de Hermetia illucens
(L.) (Diptera, Stratiomyidae).
Resumen
El objetivo de este captulo fue estudiar y determinar diferencias

morfolgicas en las fases larvarias, as como analizar la composicin de
hidrocarburos cuticulares con el fin de poder diferenciar los distintos estadios
preimaginales de H. illucens.
Entre los resultados obtenidos, destacan las diferencias en el tamao de la

cpsula ceflica y de los espirculos posteriores, as como el nmero de aberturas
espiraculares que permiten diferenciar los estadios larvarios. En el caso de los
espirculos posteriores, es la primera vez que estas estructuras se utilizan para y
diferenciar los cinco estadios larvales de H. illucens. Por otro lado, el anlisis
cuticular permiti observar cambios en la composicin de hidrocarburos
cuticulares con la edad de las larvas. Se determinaron varios picos durante el
anlisis, lo que indica que la cantidad de estas sustancias puede aportar
informacin muy til sobre la edad de la larva. Aunque estos picos no han sido
asociados al componente, sern analizados en detalle en futuros trabajos
mediante el uso de patrones.
Tanto las caracterizacin morfolgica como cuticular pueden ser

herramientas forenses para su aplicacin en la Entomologa forense,
concretamente en el clculo del intervalo postmortem, as como en el control del
proceso de cra masiva de esta especie.
- 31 -
Captulo I
1. Introduccin
La familia Stratiomyidae incluye 12 subfamilias (Parhadrestriinae,

Chiromyzinae, Beridinae, Sarginae, Raphiocerinae, Clitelariinae,
Chrysochlorininae, Hermetiinae, Stratiomyinae, Antissinae, Nemotelinae y
Pachygastrinae) con ms de 2650 especies repartidas en 375 gneros (Woodley,
2001).
Las larvas, asociadas normalmente a materia orgnica vegetal o animal en

descomposicin (Pujol-Luz et al., 2004), presentan una gran diversidad de
formas y tamaos, pudiendo ser terrestres, acuticas o semiacuticas, y
diferencindose por variaciones en la coloracin y la quetotaxia (McFadden,
1967). Hermetia illucens es una especie de la familia Stratiomyidae que puede
ser identificada por la combinacin de las siguientes caractersticas: cabeza
comprimida cuyo largo supera su anchura, quetotaxia dorsal y ventral de la
capsula ceflica, de los tres segmentos torcicos y de los ocho segmentos
abdominales, presencia del parche esternal en el sexto segmento abdominal y
morfologa de los espirculos anteriores y posteriores (Rozkon, 1982).
En la subfamilia Hermetiinae la especie Hermetia illucens (Linneaus,

1758), presenta la mayor distribucin geogrfica dentro del gnero Hermetia
(Woodley, 2001). Es una especie neotropical cuya presencia en Europa se ha
constatado en diversos puntos de la cuenca mediterrnea. Esta especie se registr
en Malta en 1926 (Lindner, 1936) y desde entonces se ha capturado en Albania,
Croacia, Francia, Italia, sur de Suiza, Portugal y Espaa (Ustuner et al., 2003;
Rozkosny & Knutson, 2007; Martnez-Snchez et al., 2011). Hermetia illucens
se registr por primera vez Espaa en 1954 (Peris, 1962). Las larvas son
saprfagas, pasando por cinco estadios larvarios ms la prepupa, determinados
difcilmente por las medidas de la cpsula ceflica (May, 1961). De hecho en la
- 32 -
actualidad es complicado determinar la edad de la larva, atendiendo a esta

caracterstica, puesto que podra variar en funcin de la dieta, temperatura, etc.
En los ltimos aos, ha tomado protagonismo en la determinacin de la

edad de los estados preimaginales las tcnicas de anlisis cuticular (Amendt et
al., 2004). Las larvas estn constituidas por una fuerte cutcula formada por una
mezcla de lpidos diversos, entre ellos hidrocarburos insaturados y saturados,
cidos grasos libres, alcoholes libres, glicridos, esteroles y aldehdos (Lockey,
1988; Hackman, 1984; De Renobales de et al., 1988; Howard, 1993).
Los hidrocarburos cuticulares son un componente vital de la cutcula de

los insectos durante los estados inmaduros como en fase adulta y poseen diversas
funciones como por ejemplo:
Proporcionar impermeabilizacin a los estados preimaginales y los

adultos (Hadley, 1981, Blomquist et al., 1993). Tambin forman
parte de los oocitos, formando una barrera protectora para el
embrin y los primeros estadios larvarios (Gu et al., 1995).
Actuar como mensajeros qumicos entre insectos (Howard, 1993) o
entre insectos y plantas permitiendo las interacciones trficas.
Como ejemplo, la composicin qumica de la superficie de las
plantas, as como los hidrocarburos de la cutcula de los insectos
que sobre ellas se desarrollan determinan la aceptabilidad de los
insectos fitfagos y de los grupos parasitoides (Espelie & Payne,
1991).
A pesar de los grandes avances de los ltimos aos en la caracterizacin

qumica y biosntesis de hidrocarburos (Blomquist et al., 1987; Lockey 1988;
Blomquist et al., 1993; Nelson & Blomquist, 1995), el almacenamiento, la
- 33 -
Captulo I
movilizacin, el transporte y la caracterizacin de hidrocarburos, han recibido

poca atencin.
La sntesis de hidrocarburos se ha estudiado en varios insectos

holometbolos, en particular en Lepidoptera, Coleoptera, y Diptera. Las fases
inmaduras sintetizan hidrocarburos mientras se alimentan y los almacenan para
su uso en etapas posteriores de desarrollo. En Lepidoptera, la sntesis de
hidrocarburos cuticular de las larvas se correlaciona con el perodo de
alimentacin activa, seguido por un cese de este proceso durante la fase de la
crislida (Dwyer et al., 1986; Guo & Blomquist, 1991). En insectos
hemimetbolos, como los Orthoptera, tambin se observa un patrn dependiente
de la edad en la sntesis de hidrocarburos (Cripps et al., 1988). En las hembras de
Blattella germanica (Blatodea), la sntesis de hidrocarburos se relaciona con el
ciclo gonotrfico, siendo alta durante la fase de alimentacin, pero baja durante
el perodo de ayuno que se corresponde con la puesta de la ooteca (Schal et al.,
1994). Por otro lado, dado que la estructura de la cutcula de insectos cambia con
la edad de los mismos (Zhu et al., 2006), su anlisis qumico puede ser utilizado
como mtodo de datacin de la edad del los estadios preimaginales, lo que tiene
un gran inters aplicado en diversos mbitos, como por ejemplo su uso en el
control de calidad de biofbricas de produccin masiva de insectos o su empleo
en entomologa forense. En este ltimo caso la adecuada estimacin de la edad
de la larva reviste de especial importancia para el clculo del intervalo
postmorten, especialmente en el caso de los dpteros (Goff & Flynn, 1991).
El anlisis de los lpidos cuticulares mediante cromatografa de gases y

espectrometra de masas, demuestran que existen distintas composiciones de
hidrocarburos no slo entre diferentes especies, sino adems entre sexos y en las
diferentes etapas del ciclo de vida de los insectos (Lockey, 1991). Incluso se han
- 34 -
encontrado diferencias cualitativas en la composicin cuticular en poblaciones

con diferentes distribuciones geogrficas, p.ej. en Blattodea: Blattella lituricollis
(Brenner et al., 1993), o en Diptera: Phormia regina (Byrne, 1995) y Chrysomya
bezziana (Brown et al., 1998).
Los estudios sobre la composicin de hidrocarburos en la cutcula de

dpteros se iniciaron en el gnero Drosophila hace aproximadamente 25 aos
(Jallon & David, 1987). Sin embargo, muy pocos estudios se han llevado a cabo
en las diferentes etapas de desarrollo de estos insectos (Goodrich, 1970;
Hebanowska et al., 1990; Espelie & Payne, 1991; Howard et al., 1995).
El objetivo de este captulo es profundizar sobre las caractersticas

morfolgicas larvarias de H. illucens, haciendo especial hincapi en la quetotaxia
y las estructuras espiraculares cuyas diferencias pudieran utilizarse para el
diagnosis de los distintos estadios larvales. Por otro lado, se buscaron cambios en
la composicin cuticular de las larvas de diferentes edades, a nivel cualitativo, es
decir sin determinar el componente exacto pero determinando su abundancia y
analizando su variacin durante el periodo de desarrollo larvario.
2. Material y Mtodos
Los especmenes de H. illucens utilizados fueron recolectados de una

colonia de laboratorio mantenida desde 2008 en las instalaciones de la
Universidad de Alicante bajo condiciones constantes (255 C, 5010 %HR, luz
natural). La colonia se origin a partir de pupas obtenidas comercialmente
(Empresa de Recursos Insect Science, Georgia, EEUU).
- 35 -
Captulo I
2.1. Caracterizacin morfolgica de las larvas
Para el estudio de la morfologa externa larval, se recolectaron huevos que

fueron transferidos a una mezcla homognea de pienso de gallina ponedora y
agua. Posteriormente se ubicaron en una cmara de cra (25 C, 60 %HR, 12:12
L:D) hasta la emergencia de las larvas. Diariamente se muestrearon 10 larvas
(24hs), hasta la obtencin de las primeras prepupas. Las prepupas o larvas de
sexto estadio no se analizaron ya que presentan estructuras que permiten
reconocer fcilmente su estadio (Rozkosny, 1982). Para su preservacin, las
larvas fueron previamente sumergidas en agua destilada y hervidas a 70-80C
aproximadamente durante cinco minutos. Seguidamente fueron transferidas a
recipientes hermticos debidamente etiquetados conteniendo alcohol etlico al
70%.
Para determinar posibles diferencias en la morfologa, en las larvas de

cada da se analizaron las diferentes estructuras como la quetotaxia, la
morfologa de los espirculos anteriores y posteriores, la morfologa de la
capsula ceflica, el largo y ancho de la capsula ceflica, la hendidura anal y la
disposicin de sensilios, papilas o espinas a lo largo de los segmentos torcicos y
abdominales. Para realizar este anlisis se utiliz microscopia electrnica y
ptica de los Servicios Generales de Investigacin de la Universidad de Alicante.
Se emple la tcnica de cryo-scanning, donde las larvas son congeladas con
nitrgeno lquido durante 3-4 minutos. Posteriormente, la muestra congelada es
transferida a la unidad de crio del microscopio electrnico de barrido (SEM)
S3000N Hitachi, donde se lleva a cabo la sublimacin de la muestra de -150C a
-90 C. Una vez eliminado el hielo superficial de la muestra se realiza el
recubrimiento metlico con oro, llamado mtodo de sputtering y se procede a
realizar las imgenes. Los espirculos posteriores, previamente extrados y
- 36 -
montados en un portaobjetos con glicerina, fueron observados y fotografiados

con microscopia ptica (Nikon, Eclipse E200) con cmara de adquisicin de
imgenes marca Infinity 1. El anlisis fue completado con fotografas tomadas en
el laboratorio de Dpteros del CIBIO (cmara Leica HD 0,5x en lupa Leica
IC80Hd).
Las larvas tienen forma esencialmente alargada (0,85 a 30 mm) con

extremo anterior ahusado y posterior redondeado (Figura 1A). El tegumento es
blanquecino a ms oscuro y fuertemente esclerotizado segn la edad, con una
apariencia de panal o de mosaico cuando se ve bajo un aumento moderado,
debido al depsito cuticular de carbonato clcico (Figura 1B) (Mller, 1925).
El cuerpo se divide en tres regiones (Figura 1A), la capsula ceflica (CC),

tres segmentos torcicos (ST) y ocho segmentos abdominales (SA). Los
segmentos del cuerpo son ms anchos que largos, y aplanados
dorsoventralmente. La diferencia entre segmentos torcicos y abdominales se
encuentra en la quetotaxia.
- 37 -
Captulo I
Lista de Abreviaturas utilizadas en las figuras
a=antena abe=aberturas espiraculares Ad=setas anterodorsales

am=rea molar an=ano An=setas anales
cae=cmara espiracular CC=cpsula ceflica ce=cicatrz estigmtica
Cf=setas clipeofrontales cm/mx=complejo mandbulo-maxilar cmx=cepillos maxilares
D=setas dorsales DL=setas dorsolaterales esa=espinas anales
ep=espirculos
ea=espirculos anteriores ecf=esclerito clpeofrontal
posteriores
esl=esclerito lateral hea=hendiduras espiraculares L=setas laterales
Lb=setas labrales lbr=labro lg=lbulos genales
m=mandbula mx=maxila O=ojos
Pa=setas preanales pae=parche esternal plg=pliegue
plv=placa ventral pmx=palpos maxilares prm=prementum
Pv=setas posteroventrales SA=segmentos abdominales Sa=segmento anal
sec=sensilios
se=sensilios smx=setas maxilares
campaniformes
ve=vestigios
ST=segmentos torcicos V=setas ventrales
espiraculares
VL=setas ventrolaterales
- 38 -
Cpsula ceflica
I
Segmentos
II
torcicos
III
Segmentos
abdominales 5
6
B
8
A
Figura 1. Apariencia general de la larva de Hermetia illucens mostrando la divisin de los

segmentos torcicos, abdominales y la capsula ceflica de la cara dorsal (A). Detalle de la
cutcula (B).
Cpsula ceflica
La cabeza es estrecha y larga, fuertemente esclerotizada y puede ser

retrada dentro del trax. Dorsalmente se compone de un esclerito dorsomedial,
el clipeo o clipeofrontal (ecf), y un par de escleritos laterales (esl), los cuales por
lo general cubren la mayor parte de la cabeza hasta la regin ventrolateral (Figura
2A). Las antenas (a) en posicin anterolateral estn formadas por tres segmentos
- 39 -
Captulo I
generalmente conspicuos, el primero o basal presenta 3 sensilios campaniformes

(sec) distribuidos homogneamente en la base (Figuras 2A, B). En posicin
lateral encontramos un par de ojos simples u ocelos (o) (Figuras 2A, C).
En la regin anterior, el clpeo se contina hacia delante conformando el

labro (lbr) estrecho y cnico (Figuras 2A, E), a cuyos lados se sita el complejo
mandbulo-maxilar (cm/mx) el cual se continua ventralmente (Figura 2D). ste
es el nico apndice bucal y se forma por la fusin de las mandbulas y maxilas
originales. A lo largo del borde interno anteroventral de los escleritos laterales se
observan los lbulos genales (lg), estrechos y coriceos (Figura 2E). Cada
complejo mandbulo-maxilar consta de una parte basal (mandbula original, m) y
una parte apical (maxila original, mx). Cada maxila presenta un palpo maxilar
(pmx) (Figura 2F,G) y una serie de setas y cepillos de barrido, denominados
cepillos maxilares (cmx) utilizados en la cavidad oral para la alimentacin
(Figura 2F). Cerca de la base del palpo maxilar se forma una agrupacin de setas
(setas maxilares, smx) poco desarrolladas (Figura 2F). Posteriormente en la
superficie ventral existe un rea molar muy desarrollada (am) (Figura 2H). En la
regin basal ventral, la placa ventral (plv), se extiende anteriormente en dos
proyecciones las cuales estn conectadas en la zona media a un estrecho
esclerito, el prementum (prm) (Figura 2D).
Quetotaxia: En la regin dorsal de la cpsula ceflica hay 4 pares de setas

dorsales, 2 pares llamadas setas labrales (Lb), y los otros 2 pares son las clipeo-
frontales (Cf). Adems existe 1 par dorsolateral (DL) detrs de las prominencias
oculares (Figura 3A). En la regin ventral hay 3 pares de setas ventrolaterales
(VL) y 3 pares ventrales (V) (Figura 3B).
- 40 -
A lbr B
ecf
esl
o
sec
C D
cm/mx
plv
prm
Figura 2. Morfologa de la cpsula ceflica de la larva de Hermetia illucens. Vista dorsal de la

cpsula ceflica (CC) (A). Detalle de la antena (a) (B). Detalle del ojo (o) (C). Vista ventral de
la CC (D). Esclerito lateral (esl), esclerito clpeofrontal (ecf), labro (lbr), sensilios
campaniformes (sec), placa ventral (plv), prementum (prm).
- 41 -
Captulo I
E F
lbr
cmx pmx
smx
lg
G H
am
Figura 2. Continuacin. Detalle del complejo mandbulo/maxilar (cm/mx) (E). Detalle de la

maxila (mx) (F). Detalle de los palpos maxilares (pmx) (G). Detalle de la rea molar (am) (H).
Lbulos genales (lg), cepillos maxilares (cmx), setas maxilares (smx).
- 42 -
VISTA DORSAL 2 pares de setas VISTA VENTRAL

labrales
3 pares de setas
ventrales
2 pares de setas
clipeofrontales
3 pares de setas
ventrolaterales
A B
1 par de setas
dorsolaterales
Figura 3. Quetotaxia de la cpsula ceflica de la cara dorsal (A) y ventral (B) del ltimo estadio
larval de Hermetia illucens (modificado de Rozkosny, 1982).
Trax
Se encuentra formado por 3 segmentos (I a III). Dorsalmente los

segmentos son densamente pilosos con varias hileras de pequeos sensilios (se)
bien desarrollados en la regin anterior de los segmentos II y III (Figuras 4A y
5A). El primer segmento se caracteriza por presentar los prominentes espirculos
anteriores (ea) (Figuras 4A, B y 5A), dispuestos lateralmente. Cada uno est
formado por una placa esclerotizada en cuyo centro se forma el rea estigmtica
de forma acorazonada con 2 hendiduras espiraculares en forma de V (hea) y en
su base la cicatriz estigmtica (ce) (Figura 4B). A ambos lados del tercer
segmento, ubicadas dorsolateralmente, existen unas estructuras pequeas y
redondas, los que se conocen como vestigios espiraculares (ve), probablemente
- 43 -
Captulo I
no funcionales (Figura 5A). Anteroventralmente, los segmentos II y III se

caracterizan por poseer abundantes y pequeos sensilios (se) (Figura 4C y 5B).
Quetotaxia: Los tres segmentos torcicos (Figura 6A) presentan 3 pares de setas
dorsales (D) y 1 par de setas dorsolaterales (DL). En el primer segmento torcico
existe adems 2 pares de setas anterodorsales (Ad). En la parte ventral se
presenta 1 par de setas ventrolaterales (VL) y 2 pares de setas ventrales (V)
(Figura 6B).
- 44 -
A B
ea
se hea
ce
se
Figura 4. Morfologa de los segmentos torcicos. Vista dorsal de los segmentos I, II y III (A).
Espirculo anterior (ea) (B). Vista ventral de los segmentos I, II y III (D). Sensilios (se),
hendiduras espiraculares (hae), cicatriz estigmtica (ce).
- 45 -
Captulo I
ea
se
ve
se
Figura 5. Vista dorsal (A) y ventral (B) de los segmentos torcicos de la larva de Hermetia
illucens. Detalle de los espirculos anteriores (ea), los sensilios (se) y los vestigios espiraculares
(ve).
- 46 -
VISTA DORSAL VISTA VENTRAL
2 pares de setas
anterodorsales
1 par de setas
I ventrolaterales
1 par de setas
dorsolaterales
Segmentos
torcicos II
3 pares de setas 2 pares de setas
dorsales ventrales
III
1 par de setas
ventrolaterales
1
1 par de setas
dorsolaterales 3 pares de setas
ventrales
2 3 pares de setas
dorsales
2 pares de setas
laterales
4
Segmentos
abdominales
5
2 pares de setas
6 ventrales
1 par de setas
anales
7 1 par de setas
dorsales 2 pares de setas
preanales
8
2 pares de setas
dorsalaterales 2 pares de setas
A B
posteroventrales
Figura 6. Quetotaxia dorsal (A) y ventral (B) de los segmentos torcicos y abdominales del
cuerpo del ltimo estadio larval de Hermetia illucens (modificado de Rozkosny, 1982).
- 47 -
Captulo I
Abdomen
El abdomen est formado por 8 segmentos. Dorsalmente los segmentos 1

al 7, estn formados por placas, ms o menos rectangulares cubiertas por
numerosas y pequeas setas (Figura 7A). Anteriormente, en cada placa existe una
hilera de sensilios (se) en forma de espinas (Figuras 7A y 8A). Los segmentos 1 a
7 se caracterizan por presentar a ambos lados vestigios espiraculares (ve), igual
que los observados en el segmento torcico III (Figura 8A). Ventralmente, los
segmentos se caracterizan por la escasa presencia de pequeas setas y por el
desarrollo de una hilera de fuertes sensilios (se) en la regin anterior de cada
segmento (Figura 7B).
De los ocho segmentos abdominales, el ltimo o segmento anal (Sa) tiene

una forma redondeada (Figuras 7C y 8A). En su pice existe una abertura
rodeada de pequeas setas que conduce a la cmara espiracular (cae) (Figuras
7C, D) en cuyo interior se encuentran en posicin dorsal un par de espirculos
posteriores (ep). Estos espirculos estn formados por numerosas aberturas
espiraculares (abe) dispuestas radialmente sobre la cicatriz ecdisial (ce) (Figura
8A). El ano (an) aparece como una hendidura longitudinal ventral en la mitad
ventral del segmento anal y sus bordes aparecen festoneadas por espinas cnicas
(es) cortas y fuertes (Figuras 7E, F y 8B). Por encima de la hendidura anal existe
un marcado pliegue convexo (plg) (Figura 7E).
Otra estructura abdominal interesante es el llamado parche esternal (pae),

presente en la zona medio ventral del segmento 6 (Figuras 7G, H y 8B). Es una
zona desprovista de setas tricoides, con forma alargada oval, donde se distingue
la presencia de facetas cuticulares notablemente pequeas (rea cuticular de
glndulas especializadas) y con una coloracin diferente del resto del segmento.
- 48 -
A B
se se
C D
cae
cae
Figura 7. Morfologa de los segmentos abdominales de una larva de Hermetia illucens. Vista
dorsal (A). Vista ventral (B). Vista dorsal del segmento anal (C). Detalle de la cmara
espiracular (cae) (D). Sensilios (se).
Quetotaxia: Los segmentos abdominales 1 al 7 tienen setas muy similares,

aunque stas a menudo se hacen ms largas y fuertes caudalmente. Hay 3 pares
de setas dorsales (D) dispuestas como en los segmentos torcicos, adems de 1
par dorsolateral (DL), 2 pares laterales, que diferencian estos segmentos de los
abdominales (Figura 6A). Ventralmente aparece 1 par ventrolateral (VL) y 3
pares de setas ventrales (V) (Figura 6B). El ltimo segmento abdominal o
segmento anal muestra un slo par de setas dorsales (D) ms o menos
- 49 -
Captulo I
desarrolladas y dos pares de setas dorsolaterales (DL) (Figura 6A); y en la regin

ventral 2 pares ventrales (V), 2 pares posteroventrales (Pv), 1 par de setas anales
(An) y 2 pares de setas preanales (Pa), (Figura 6B) (modificado de Rozkosny,
1982; Woodley, 2009).
E F
plg
an
esa
G H
pae
Figura 7. Continuacin. Vista ventral del segmento anal (E). Detalle del ano (an) (F). Detalle
del parche esternal (pae) (G, H). Pliegue (plg) y espinas anales (esa).
- 50 -
se
ep
ve
abe
ce
cae
pae
an
Figura 8. Vista dorsal (A) y ventral (B) de los ltimos tres segmentos abdominales de la larva
de Hermetia illucens. Detalle de los espirculos posteriores (ep), los sensilios (se), los vestigios
espiraculares (ve), el parche esternal (pae) y ano (an). Camara espiracular (cae), aberturas
espiraculares (abe) y cicatriz ecdisial (ce).
- 51 -
Captulo I
2.2. Cuantificacin de hidrocarburos cuticulares
Para la extraccin de los hidrocarburos se utiliz hexano al 95% como

disolvente orgnico. El material de vidrio utilizado fue enjuagado con este
disolvente antes de ser utilizado. La metodologa seguida fue la siguiente: cada
espcimen era cubierto durante 15 minutos con 200l de hexano a temperatura
ambiente dentro de una campana extractora. Trascurrido este tiempo el
disolvente se evapora casi completamente. Posteriormente, los extractos crudos y
secos que permanecen en el vial son disueltos nuevamente con 20l de
disolvente para su posterior anlisis mediante cromatografa de gases-
espectrometra de masas (CG-EM). Este proceso se replic 48 veces (3 rplicas
para cada una de las larvas diarias).
La CG-EM es una tcnica que combina las caractersticas de la

cromatografa gas-lquida y espectrometra de masas para identificar diferentes
sustancias en una muestra de ensayo. Para la realizacin de anlisis cualitativo de
los hidrocarburos cuticulares se utiliz un cromatgrafo de gases Hewlett
Packard 6890 equipado con un capilar de 30m, una columna HP-5 y un detector
de ionizacin de llama (FID). Se inyectaron 2l de los hidrocarburos cuticulares
extrados de las diferentes muestras (n=48) y los picos cromatogrficos
resultantes se integraron mediante software (Hewlett Packard) para su
identificacin con un cromatgrafo de gases Agilent 6890N conectado a un
detector de masas Agilent 5973 selectivo. Los anlisis se realizaron en los
servicios generales de investigacin de la Universidad de Alicante.
- 52 -
2.3. Anlisis estadstico
Para determinar las posibles diferencias diarias entre el tamao de la

capsula ceflica de las larvas o el tamao del cuerpo (capsula ceflica=largo x
ancho de capsula; cuerpo=largo x ancho de cuerpo de larva), asi como para
realizar las comparaciones estadsticas de la abundancia de hidrocarburos de
todas las edades larvales, se utiliz el test no paramtrico de Kruskal-Wallis (H),
ya que los datos analizados no pasaron el test de normalidad Kolmogorov-
Smirnov. El programa utilizado fue SigmaStat (versin 3.5 para Windows) y los
valores de p superior a 0,05 se descartaron.
3. Resultados
3.1. Anlisis morfolgico

En el anlisis de microscopia electrnica, debido al gran tamao de las
larvas, a partir del dcimo da solo se estudi la cpsula ceflica, los espirculos
anteriores y el segmento anal; el resto de estructuras fue analizado mediante lupa
binocular ptica, a excepcin de los espirculos posteriores que fueron
analizados diariamente mediante microscopia ptica. Los resultados obtenidos no
indicaron cambios en la quetotaxia de la cpsula ceflica en funcin de la edad de
las larvas (Figura 3). En cuanto a la quetotaxia de los segmentos torcicos y
abdominales tampoco se encontraron diferencias con respecto a la presencia o
ausencia de setas (Figura 6). S que se aprecian cambios en el parche esternal, los
espirculos posteriores y el tamao de la capsula ceflica. Al comparar el parche
esternal en los diversos especmenes muestreados se observa un aumento
marcado y progresivo de su tamao (Figura 9).
- 53 -
Captulo I
Los espirculos posteriores fueron analizados a partir del quinto da,

debido a la imposiblidad de aislarlos en larvas de menor edad por su pequeo
tamao (0,85 mm de longitud). A partir del octavo da (Figura 10) se diferencia
internamente en cada uno de los espirculos una membrana que podra
interpretarse como la sutura ecdisial (sue). Tambin se observa un aumento
progresivo del dimetro de los espirculos, as como del tamao del peritrema
(pm). A medida que aumenta la edad de las larvas, se da un aumento progresivo
en el nmero de aberturas espiraculares (abe), ajustandose a una lnea de
tendencia logartmica (Figura 11) (Tabla 1).
- 54 -
A B
pae
C D
pae
E F
pae
Figura 9. Parche esternal (pae) de Hermetia illucens de las larvas muestreadas el da 4 (A, B),
el da 6 (C, D) y el da 9 (E, F) del experimento.
- 55 -
Captulo I
B
pm
sue
abe
Figura 10. Morfologa de los espirculos posteriores (x40) de una larva de 6 das de edad (A) y
de una larva de 8 das de edad (x40) (B) de Hermetia illucens. Sutura ecdisial (sue), aberturas
espiraculares (abe), peritrema (pm).
- 56 -
80
70
N de aberturas espiraculares
y = 27,207ln(x) + 7,6005
60 R = 0,9077
50
40
30
20
10
0
5 6 7 8 9 10 11 12 13 14 15 16
Edad larval (das)
Figura 11. Nmero de aberturas espiraculares de los espirculos posteriores de larvas de

Hermetia illucens de 5 a 16 das de edad.
Finalmente, tambin el tamao de la capsula ceflica se incrementa con el

tamao y la edad de la larva (Figura 12A, B) (Tabla1). Al analizar el tamao
(largo x ancho) de la cpsula ceflica y del cuerpo de los especmenes
muestreados, se encontraron diferencias significativas (H=145,454; p0,001;
H=155,63; p0,001; respectivamente).
- 57 -
Captulo I
A
4
3,5
Longitud x anchura (mm)
2,5
1,5
0,5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Das muestreados
B
140
120
Longitud x anchura (mm)
100
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Das muestreados
Figura 12. Largo por ancho (mm) de la cpsula ceflica (A) y del cuerpo de la larva (B) de
Hermetia illucens durante los 16 das muestreados.
- 58 -
Tabla 1. Caractersticas potenciales para diferenciar los distintos estadios (L-I, L-II, L-III, L-IV,
L-V). Longitud de la capsula ceflica (mediaDS), nmero de aberturas espiraculares y tamao
del espirculo posterior de las larvas de 1 a 16 das de edad [*: medidas observadas por May
(1961) y Oliveira-Costa (2003); **: medidas obtenidas en este estudio].
Dimetro
Cpsula ceflica Cpsula ceflica N aberturas espirculo
Edad y estadio
(mm) * (mm) ** espiraculares ** posterior
(micras)**
Dia 1 (L-I) 0,200,0103 --- ---
Da 2 (L-I) 0,280 0,240,0063 --- ---
Da 3 (L-I) 0,270,0074 --- ---
Da 4 (L-II) 0,420,0191 --- ---
0,460
Da 5 (L-II) 0,670,0183 14 86,67
Da 6 (L-III) 1,040,0421 15 86,67
0,680,09
Da 7 (L-III) 1,10,0294 30 146,67320
Da 8 (L-IV) 1,490,1066 52 320
Da 9 (L-IV) 1,640,0644 53 340
Da 10 (L-IV) 1,760,0777 69 386,67
1,260,09
Da 11 (L-IV) 1,840,0883 60 406,67
Da 12 (L-IV) 1,850,0782 61 406,67
Da 13 (L-IV) 1,940,0606 63 413,33
Da 14 (L-V) 1,990,0443 72 540
Da 15 (L-V) 2,020,36 2,040,0681 73 540
Da 16 (L-V) 2,080,2226 73 540
- 59 -
Captulo I
3.2. Anlisis de hidrocarburos cuticulares
La composicin cualitativa de los diferentes hidrocarburos cuticulares, as

como los distintos cromatogramas de las larvas de H. illucens analizadas durante
los 16 das de experimento, se muestran en la figura 13. Se observa que a medida
que aumenta la edad de las larvas aumenta la abundancia de los diferentes
compuestos hidrocarbonados (H=212,584; p0,001), siendo significativamente
mayor esta diversificacin en las larvas de 7, 9 y 10 das. A los 8 minutos de
arrastre de los diferentes compuestos cuticulares aparece un pico de un
compuesto hidrocarbonado cuya abundancia aumenta progresivamente con la
edad de las larvas (H=35,87; p=0,002), siendo significativamente mayor su
abundancia en las larvas del da 16 (Figura 13) (Tabla 2). Este resultado permite
diferenciar las larvas de diferentes edades simplemente por la abundancia de este
compuesto (Tabla 2).
A medida que aumenta la edad de las larvas aumenta progresivamente la

abundancia de ciertos compuestos hidrocarbonados, destacndose algunos de
ellos que pueden utilizarse para diferenciar los distintos estadios larvales (Tabla
3).
- 60 -
Tabla 2. Abundancia media de compuestos hidrocarbonados y de los picos ms destacados de

hidrocarburos (3, 8, 9-10 y 11 minutos de retencin) en larvas de H. illucens de 1 a 16 das de
edad (*p0,05).
Abundancia Media
Edad Larva (das) del total de Tiempo de Retencin (min) Abundancia DS
hidrocarburos
3,22 105.5615.290
110.899 8,22 190.900992

1
--- ---
11,22 88.5026.516
3,28 115.9428.502
111.503 8,23 318.5361.807

2
9,45 151.8441.019
11,22 85.8721.519
3,44 222.5730
113.431 8,21 359.37414.171

3
9,45 43.1128.353
11,22 156.5238.400
3,98 229.0841.029
172.794 8,21 388.4192.389

4
--- ---
11,22 60.8860
3,97 248.9561.000
177.867 8,30 425.1563.171

5
--- ---
11,22 89.7900
3,70 201.5712.316
196.951 8,20 689.1232.871

6
--- ---
11,23 70.2220
3,99 360.8540
399.060 8,31 955.6061.414

7
9,45 995.2531.698
11,37 454.1043.433
3,50 726.9792.994
406.495
8 8,22 979.59510.058
9,65 962.4464.509
- 61 -
Captulo I
Tabla 2. Continuacin.
11,27 977.3891.218
3,56 999.4940
408.957 8,22 993.2801.414

9
9,65 974.4116.258
11,25 990.3461.646
3,35 706.5630
410.708 8,22 1.009.405527

10
9,65 535.6194.969
11,25 999.2763.212*
3,24 504.5960
8,22 1.114.527625
413.852
11
9,01 428.8643.091
--- ---
3,99 722.2953.109
414.625 8,25 129098012.832

12
--- ---
--- ---
3,94 1.265.5111.395
474.526 8,21 1.410.987931

13
--- ---
--- ---
3,99 1.496.3860
425.019 8,23 1.445.6544.306

14
9,99 448.0642.343
--- ---
--- ---
432.843 8,23 1.574.8451.352

15
9,76 192.9621.177
11,25 610.9740
3,93 1.890.9290*
446.063 8,25 2.786.0032970*

16
9,75 235.7520
11,26 1.669.4140*
- 62 -
3,0E+06 DA 2
DA 1
2,5E+06
2,0E+06
1,5E+06
1,0E+06
5,0E+05
0,0E+00
0 4 8 12 16 20 24 0 4 8 12 16 20 24
3,0E+06
DA 3 DA 4
2,5E+06
2,0E+06
1,5E+06
1,0E+06
5,0E+05
0,0E+00
ABUNDANCIA
0 4 8 12 16 20 24 0 4 8 12 16 20 24
3,0E+06 DA 6
DA 5
2,5E+06
2,0E+06
1,5E+06
1,0E+06
5,0E+05
0,0E+00
0 4 8 12 16 20 24 0 4 8 12 16 20 24
3,0E+06
DA 7 DA 8
2,5E+06
2,0E+06
1,5E+06
1,0E+06
5,0E+05
0,0E+00
0 4 8 12 16 20 24 0 4 8 12 16 20 24
TIEMPO DE ARRASTRE (MIN)
Figura 13. Cromatogramas (media) de los hidrocarburos cuticulares de larvas de Hermetia
illucens muestreadas del da 1 al da 8.
- 63 -
Captulo I
3,0E+06
DA 9 DA 10
2,5E+06
2,0E+06
1,5E+06
1,0E+06
5,0E+05
0,0E+00
0 4 8 12 16 20 24 0 4 8 12 16 20 24
3,0E+06
DA 11 DA 12
2,5E+06
2,0E+06
1,5E+06
1,0E+06
5,0E+05
ABUNDANCIA
0,0E+00
0 4 8 12 16 20 24 0 4 8 12 16 20 24
3,0E+06 DA 14
DA 13
2,5E+06
2,0E+06
1,5E+06
1,0E+06
5,0E+05
0,0E+00
0 4 8 12 16 20 24 0 4 8 12 16 20 24
3,0E+06
DA 15 DA 16
2,5E+06
2,0E+06
1,5E+06
1,0E+06
5,0E+05
0,0E+00
0 4 8 12 16 20 24 0 4 8 12 16 20 24
TIEMPO DE ARRASTRE (MIN)
Figura 13 (continuacin). Cromatogramas (media) de los hidrocarburos cuticulares de larvas
de Hermetia illucens muestreadas del da 9 al da 16.
- 64 -
4. Discusin
Hermetia illucens parece ser una especie euriterma, es decir que puede
tolerar temperaturas extremas, y puede vivir en diferentes tipos de medios. Como
se ha visto las larvas son polfagas y se han criado a partir de diversos productos
orgnicos, frutas y verduras en descomposicin, cadveres animales y humanos y
hasta en letrinas (Rozkon, 1982). Todas estas caractersticas hacen que se
considere una especie de gran importancia econmica que necesita de ms
estudios biolgicos y morfolgicos, para su ptima utilizacin.
A pesar de que existen varias caractersticas que determinan la morfologa

de la especie, solo el tamao de la cpsula ceflica puede considerarse como
caracterstica que contribuye a determinar los seis estadios larvales) (May, 1961;
Oliveira-Costa, 2003). En este trabajo se aportan dos nuevas estructuras,
espirculos posteriores y el parche esternal, que permiten la datacin de la larva
ya que aumentan el tamao progresivamente con la edad; lo mismo sucede con el
nmero de aberturas espiraculares, aumentando conforme aumenta la edad de la
larva. Sin embargo no es fcil establecer una relacin con los diferentes estadios
larvales. Los datos aqu presentados confirman que la cpsula ceflica permite
diferenciar estadios, pero tambin el tamao de los espirculos y sus aberturas
respiractorias. As la L-I dur los 3 primeros das y present una cpsula ceflica
menor de 0,30. Los espirculos posteriores fueron visibles pero no se pudieron
analizar. La L-II present 2 das de duracin (da 4-5) y la cpsula fue menor de
0,68 y mayor de 0,40, siendo los espirculos medibles con poco ms de una
docena de aberturas y difcilmente distinguibles del estadio siguiente. La L-III se
di entre el da 6 y 7. La cpsula ceflica midi sobre un 1 mm y las aberturas
espiraculares rondan un mximo de 30. El dimetro se incrementa bruscamente.
La L-IV, entre el da 8 y 13, se caracteriza por ser el periodo ms largo, con una
longitud de la cpsula ceflica entorno al 1,5-2 mm y aberturas entre 50 y 60. El
- 65 -
Captulo I
dimetro se duplica con respecto al estadio anterior. Finalmente la L-V (da 14 al

16) presenta una cpsula ceflica de 2 mm o ms y el nmero de aberturas (72-
73) y longitud de los espirculos (540 micras) se estabiliza.
Las larvas de H. illucens han sido estudiadas y/o descritas por otros
autores como Bez (1975), Rozkon (1982), Shremmer (1986) y Wontae
(2010). Sin embargo es la primera vez que los espirculos posteriores han sido
aislados, analizados y utilizados para determinar los estadios larvarios.
Estos resultados preliminares aportan informacin importante de cuales

son las estructuras en las que se necesita profundizar, ya que podran facilitar la
datacin de la larva y colaborar en los estudios de ndole forense (determinacin
del intervalo post mortem) o en estudios de cra masiva logrando una cra
sostenida y controlada.
En cuanto al anlisis de los hidrocarburos cuticulares los resultados

determina que el perfil de hidrocarburos cuticulares de las larvas de H. illucens
siguen un patrn con respecto a la edad. Por un lado, hay un aumento de la media
de su abundancia total al aumentar la edad de las larvas, y por otro lado la
composicin de picos correspondientes a compuestos de mxima abundancia
diaria, prximos al 3, 9 y 11 minutos de retencin, y la abundancia propia del
pico a los 8 minutos, pueden ser utilizados como marcadores fiables de la edad
de las larvas. Sin embargo, los resultados no indican en muchos casos diferencias
significativas. Resultados similares se encontraron en larvas de otros dpteros
(Zhu et al., 2006), as como en algunos insectos sociales (Monnin & Peeters,
1999) donde los hidrocarburos cuticulares diferenciados por la edad podran
actuar como marcador importante en la alimentacin y en la distribucin de
edades de la colonia (Wagner et al., 1998).
- 66 -
Hermetia illucens suele intervenir como necrfago secundario en estudios

de entomologa forense, pudiendo aportar informacin importante en el clculo
del intervalo postmortem (Catts & Haskell, 1990; Lord et al., 1994; Pujol et al.,
2008). Esta estimacin depende de la tasa de desarrollo de cada especie, que a su
vez depende de la temperatura y humedad del entorno. Para determinar la edad
de las larvas se utiliza con frecuencia la longitud de las mismas; sin embargo, la
longitud no es un criterio til en la fase de post-alimentacin larvaria pudiendo
representar una fuente de distorsin entre el crecimiento experimentado durante
los primeros estadios larvales y el ltimo o prepupa (Greenberg, 1991). Por ello,
el anlisis de hidrocarburos cuticulares puede ser un mtodo til para determinar
la edad de las larvas de H. illucens, debido a los cambios en las proporciones de
los picos de abundancia de los diferentes compuestos (Zhu et al., 2006). Este
hecho puede ser utilizado tambin como medida de control de calidad de un
sistema de produccin masiva de insectos, especialmente en el caso de H.
illucens ya que puede permanecer durante largos periodos de tiempo sin recibir
alimento, de ah una de sus denominaciones comerciales ms comunes gusano
Phoenix.
Como conclusin, los resultados de este estudio demuestran que el tamao

de la cpsula ceflica, el tamao de los espirculos posteriores, el nmero de
aberturas espiraculares y la composicin de hidrocarburos cuticulares en larvas
de H. illucens cambia gradualmente con la edad. Sin olvidar que otros factores
distintos a la edad pueden influir en estas caractersticas, poseen un gran
potencial para su empleo en la datacin de la edad de las larvas, aunque son
necesarios estudios complementarios sobre su variabilidad en funcin de la
temperatura o tipo de alimentacin.
- 67 -
Captulo I
Este estudio preliminar dar paso a futuros trabajos, entre ellos, aquellos
estudios que determinen cuales son los componentes especficos de los picos 3,
8, 9 y 11 y su variacin no slo a lo largo de la vida larvaria sino tambin de la
pupa y el adulto, o tambin su variacin en funcin de diferentes variables.
- 68 -
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- 75 -
Captulo I
- 76 -
Captulo II
Growing curves of Hermetia illucens
Growing curves of the Black Soldier Fly, Hermetia

illucens (Diptera: Stratiomyidae) in two different
larvae media.
Abstract
Recently Hermetia illucens (Linnaeus, 1758) (Diptera: Stratiomyidae) has

been found in human corpses in Europe then, this species need to be considered
as a forensic indicator to estimate the post-mortem interval (PMI). The species
also shown a high potential related with its mass-production related with its use
as animal feed. Study of growing-degree days is a key factor on applied research
in entomology.
Life cycle of this species has been studied at three constant temperatures:
25 C, 30 C and 35 C. Larvae were measured and weighed, and individualized
pupae were weighed until adult emergence. The variation in development time
and size of larva and pupa were recorded in two different diets: mixed meat (pig)
and hen feed. We also calculated the minimum development time, the degree-
days accumulated and isomorphic diagrams.
Gobbi, P.; Martnez-Snchez, A. & Rojo, S. 2012 (November). Growing curves of the Black Soldier
Fly, Hermetia illucens (Diptera: Stratiomyidae) in two different larvae media. Journal of Forensic
Science International. Submitted.
- 79 -
Captulo II
1. Introduction
The Black Soldier Fly (BSF), Hermetia illucens (Linnaeus, 1758)

(Diptera: Stratiomyidae) is originally a New World species, but human activity
established this species in all regions (Ustuner et al., 2003; Martnez-Snchez et
al., 2011). The BSF is a generalist detritivore species, which colonizes a wide
variety of decomposing plant and animal matter (Sheppard et al., 2002; Puyol et
al., 2008). Similar to other stratiomyids of the subfamilies Hermetiinae and
Sarginae, H. illucens can be classified as opportunistic or secondary
necrophagous and may be important for forensic entomology, especially in
estimation of the postmortem interval (PMI) (Catts & Haskell, 1990; Lord et al.,
1994; Puyol et al., 2008).
To estimate the period of time since death (PMI) two methods can be
used: calculation based on development of individual species and use of
succession studies (Centeno, 2002). In the succession of insects, BSF is
considered a late colonizer (Dunn, 1916; Lord et al., 1994, Tomberlin et al.,
2005). Both methods can be applied separately or together depending on the
analysis performed. The first method is used during the early stages of
decomposition, which involves a few species of insects, particularly flies. The
estimates are based in the degree of species development involved and their
comparison with curves growth obtained in similar climatic and geographical
conditions. The second method is used in advanced stages of decomposition, is
based on the comparison of fauna found in the body with typical faunal
succession patterns habitat where the body was found. In order to this, the
identification of species, the knowledge of their life cycles, the duration of each
stage depending and other factors abiotic data are needed to determine the PMI
(Centeno, 2002).
- 80 -
For calculating PMI is important to know how environmental factors

influence the development of insects. Within many extrinsic factors that
influence in the development of the insects are the environmental conditions such
as temperature, humidity and food. The different larval instars have different
temperature optima and limits (Howe, 1967). Most techniques for predicting an
age on insects, is based on the relationship between development rate and
temperature. One method used is the calculation of ADH (accumulated degree-
hours) or ADD (accumulated degree days), defined as the amount of heat
required by an organism to complete the various stages of development in its life
cycle (Greenberg, 1991; Goff, 1993). Therefore, knowing insects development
thresholds and data daily temperatures registered, can be calculate the minimum
time from ovo/larviposicion of the insect.
Although Hermetia illucens biology has been studied by several authors

(Dunn, 1916; Booth & Sheppard, 1984, Sheppard et al., 2002; May, 1961; Tingle
et al., 1975; Tomberlin et al., 2005) some aspects of their development, as study
of growing-degree days are unknown. For this reason, the main objective is study
will be the development of growing curves of BSF at different temperatures and
with different larval diets. These data will be useful for both forensic and other
applied research on this species.
2. Methodology
The BSF specimens used in this experiment were originated from pupae
commercially available (Insect Science Resource Company, Georgia, USA). The
adults were placed in colony cages of 3 m and kept in a glass greenhouse
module under controlled conditions (255 C, 5010 % RH and natural light).
Adults were fed sugar and water. Approximately 7 days after emergence, a
mixture of water and hen feed (500 gr diluted in 800 ml of water) was offered for
- 81 -
Captulo II
oviposition. The medium with eggs was transferred to a climatic chamber (25 C,
60 % RH, 12:12) to optimize hatching. Later, hen feed medium was supplied ad
libitum as larvae media. When larvae reach prepupal stage, they left the rearing
medium to pupate in a sand tray placed at the bottom of the container. The pupae
were filtered and transferred to the adult cages again.
Eggs in oviposition medium (12 h) were introduced into a climatic

chamber at different temperatures (25-30-35 C and 60 % RH) to be studied full
development of BSF. Eggs were observed every 12 hours until hatching, which
has considered the day of the first larva. Since pig liver produced high mortality
in the initial probes, 600 larvae (12 h) were selected and arranged in two
different diets in small pots, an optimal diet of hen feed (500 gr diluted in 800 ml
of water) and a similar carrion diet composted by a mixed of swine meat (200 gr
swine liver+200 gr bacon+200 gr swine pig lean meat). Subsequently, every diet
pot was introduced into other, with sand on the background to pupa. For each
temperature and diet 5 replicates were performed.
Each day, 10 larvae were collected from each replicate in each diet until
the first 10 pupae were observed. The larvae were weight in a precision balance
(0.0001 gr) and then boiled for 5 minutes; subsequently, its length was
measured with a calliper digital (0.01 mm) and preserved in alcohol 70 %.
Then, the first 10 pupae from each replicate were individualized, leaving at the
same temperature of the experiment and weighing daily until adults emerged,
when they were sexed. To calculate accumulated degree days (ADD) the
following formulates was using: ADD=y (t-t0) being y development time in days,
t breeding temperature (C) and t0 the minimum threshold of development of the
species, which had to be calculated by the representation of the breeding
temperature (X axis) versus 1/development time (Y axis). Finally, a diagram
isomorphic was developed to results in swine meat of H. illucens, since this
- 82 -
figure is a representation to apply in forensic entomology, mainly. In the diagram

from oviposition, larval and pupal time in each temperature was represented.
To determine the duration of the life cycle was used the minimum
duration of larvae, when the first larvae of each replicate pupated. The duration
of the pupal period was calculated from the first 10 pupae observed per replicate.
To construct the growth curves was plotted on a graph the average maximum
length and weight of 10 larvae daily measurements for each temperature.
For all statistical analyses, when data did not meet the assumption of
normality non-parametric test Kruskal-Wallis (H) and Mann-Whitney (U),
followed by Tukey test for post hoc multiple comparisons were performed. Data
were considered significant when p value was 0.05. All analysis performed
using the SigmaStat 3.5 program.
3. Results
When both diets were compared, H. illucens had higher development rate
in hen feed than in swine meat (U=50; p=0.001) (Table 1). The total life cycle of
BSF decreased when the temperature increases in hen feed (H=12.5; p=0.002)
and in swine meat (H=12.52; p=0.002). Moreover, statistically significant
differences were found in the duration of larval at different temperatures, in both
diets, and in the pupal stage in swine meat (Larva: hen feed H=12.57, swine meat
H=12.52; p=0.002; Pupa: hen feed H=5.58; p=0.061, swine meat H=8.70;
p=0.013). So, the larva and pupa stages were shorter when temperature increased,
except in pupae reared at 35 C where increased (Table 1). However significant
differences only were observed between 25 C and the rest of temperatures,
therefore the results indicate that larval stage is shorter at temperatures higher
than 25 C.
- 83 -
Captulo II
Table 1. Minimum period (meanSD) of the egg, larval and pupal stage and complete period
(mean SD) of Hermetia illucens at constant temperatures and different diets (* indicate
significant differences at p <0.05 in each diet).
T Egg Stage Larval Stage Pupal Stage Life Cycle

Diet
(C) (days) (days) (days) (days)
25 30 34.560.51* 6.790.22 44.360.42*
Hen Feed 30 30 31.20.34 6.190.34 40.390.16
35 20 20.120.39 8.421.66 30.541.70
25 30 46.220.23* 7.840.25* 57.060.18*
Swine
30 30 37.440.50 7.290.74 47.730.62
Meat
35 20 26.30.55 8.360.27 36.660.58
In order to application in Forensic Entomology, ADD and isomorphic

diagram were realized. To calculate ADD (Table 2), the minimum threshold of
development (t0) in BSF had to be calculated in hen feed and swine meat (Figure
1); in both equations of regression when y was zero, x was 3.9 and 7.3
respectively. The ADDs were higher in larvae stage, then in prepupa, pupae and
finally in egg stage (Table 2). Results indicated higher value at 30 C and the
lowest 25 C, in both diets. However differences in ADDs from stages in base to
temperature were observed (Table 2). In both diets, egg and larva stage need
more ADD than prepupa or pupa at 30C, and prepupa and pupa stages need
more ADD at 35 C. The isomorphic diagram (Figure 2), allows calculate age of
the eggs, larvae, prepupa and pupae of BSF at different temperatures between 25
to 35 C, by extrapolation.
- 84 -
hen feed swine meat

0.045
0.040 y = 0.001x - 0.0039
Development index (1/days)
0.035 R = 0.9034
0.030
0.025
0.020
y = 0.001x - 0.0073
0.015 R = 0.9713
0.010
0.005
0.000
0 10 20 30 40 50
Temperature (C)
Figure 1. Relationship between the development index and development temperature of

Hermetia illucens in hen feed and swine meat.
Table 2. Accumulated Degree Days (ADD) in every stage and prepupa install. calculated from
average data of development in different conditions (diets and temperature) and minimum
thresholds (3.9 from hen feed and 7.3 from swine meat).
Diet T (C) ADD ADD ADD ADD ADD

egg larve prepupa pupa Life cycle
25 63.3 464.2 265.02 143.48 935.95
30 78.3 522 292.32 161.56 1054.33

Hen Feed
35 62.2 311 314.73 261.86 949.79
25 53.1 566.4 251.69 138.77 1009.96
Swine 30 68.1 590.2 259.68 165.48 1083.47

Meat
35 55.4 443.2 285.31 231.57 1015.48
- 85 -
Captulo II
35
Hen Feed
34
Swine meat
33
32
Temperature (C)
31
30 A A
29
28
27
26 E L L Pr P Pr P
E
25
0 5 10 15 20 25 30 35 40 45 50 55 60
Time from oviposition (days)
Figure 2. Isomorphic diagram of Hermetia illucens showing the different morphological stages:
eggs (E), larval (L), prepupal (Pr), pupal (P) and adult (A), at 25, 30 and 35 C in swine meat
(black) and hen feed (red).
The length and weight of larvae every day was reporting. The last days in
the life of the larvae (from the 11th day or more, depend of temperature and
diets), the prepupa install begins and the length remains constant (Figure 3) and
decrease the weight seemed more or less pronounced depending on temperature
and diet (Figure 4). Both variables increase when temperature increase in the two
diets (Figures 3 to 6), at least in the first 15-20 days. With the increasing of the
length, weight increases too (Figure 5) in both diets, observed a positive
relationship between the two variables, exponential o polynomial in hen feed and
swine meat respectively.
- 86 -
35
25 C
30 30 C A
35 C
25
Larval length (mm)
20
15
10
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
Larval development time (days)
35
25 C
30 30 C B
35 C
25
Larval length (mm)
20
15
10
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Figure 3. Larval length curve of H. illucens to 25 C, 30 C and 35 C in hen feed (A) and swine
meat (B).
- 87 -
Captulo II
0.5
25 C
30 C A
0.4 35 C
Larval weight (g)
0.3
0.2
0.1
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
0.5
25 C
30 C B
0.4 35 C
Larval weight (g)
0.3
0.2
0.1
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Figure 4. Larval weight curve of H. illucens to 25 C, 30 C and 35 C in hen feed (A) and
swine meat (B).
- 88 -
0.4 0.4
A B
y = 0.0002e0.2909x
0.35 y= 0.0001e0.2897x 0.35 R = 0.8889
R = 0.8969
0.3 0.3
0.25 0.25
Weigth (g)
Weigth (g)
0.2 0.2
0.15 0.15
0.1 0.1
0.05 0.05
0 0
0 10 20 30 0 10 20 30
Length (mm) Length (mm)
Figure 5. Relationship between the weigth and the length of larvae of Hermetia illucens in hen
feed (A) and swine meat (B).
100
90
80
70
60 *
50 *
40
30
20
10
0
25 C 30 C 35 C 25 C 30 C 35 C
Hen feed Swine meat
Figure 6. Mortality percent (meanSD) at constant temperatures (* indicate differences in

treatments with p<0.05).
- 89 -
Captulo II
When adults emerged, mortality rate was obtained in both diets, being
greater in swine diet, however these differences were not significant (p=0.4)
(Figure 6). In both diets, hen feed (H=12.33; p=0.002) and swine meat
(H=10.33; p=0.006), mortality increased when temperature did, being significant
at 35 C (p<0.05). There were no differences in the weight of females or males
pupae in both diets (Table 3). When comparing, significant differences were
found in the weight of pupae reared in hen feed and swine meat at different
temperatures (H=53.54; p0.001), being lighter when larvae are rearing in swine
meat. In pupae developed in the hen feed there was a reduction of the weight of
pupae when temperature decreased (H=33.92; p0.001) being the lightest pupae
reared at 25 C (Figure 7). However in swine meat the differences between
weights were not significant at three temperatures. In both diets was observed a
decreasing in the weight with the development of pupa (Figure 7).
Table 3. Weigth (gr) of female and male pupae (meanSD) of Hermetia illucens at the different
temperatures in hen feed and swine meat.
Temperature Hen Feed Swine Meat

(C) Female Male Female Male
25 0.120.01 N=17 0.120.01 N=31 0.120.02 N=17 0.110.02 N=23
30 0.180.02 N=13 0.170.02 N=21 0.100.01 N=8 0.110.03 N=16
35 0.160.02 N=9 0.150.02 N=16 0.110.03 N=9 0.120.03 N=12
- 90 -
0.18
0.16 A
0.14
Pupal wegith (g)
0.12
0.1
0.08
0.06 25 C
30C
0.04
35C
0.02
0
1 2 3 4 5 6 7 8 9 10
0.18
0.16 B
0.14
Pupal wegith (g)
0.12
0.1
0.08
0.06 25C
0.04 30C
35C
0.02
0
1 2 3 4 5 6 7 8 9 10 11 12
Figure 7. Pupal weigth curve of H. illucens to 25 C, 30 C and 35 C in hen feed (A) and swine
meat (B).
- 91 -
Captulo II
4. Discussion
In the calculation of age or development time in BSF, an important factor

is the ambient temperature in the different stages. The BSF is very sensitive to
changes in temperature. The rate of development, as other ectothermic insects, is
directly related to temperature. As temperature increases, the rate of development
increases and durations of the individual stages in the life cycle of become
shorter. Tomberlin et al. (2009) reported a strong influence of environmental
temperature on the development of BSF immature stages reared under laboratory
conditions.
The results indicate life cycle of Hermetia illucens decreases with

increasing temperature. This occurs mainly by decreasing in the development of
larvae. However, pupae reared at 35C needed more time to complete develop.
Other authors have studied the life cycle of BSF at constant temperature but in
particular conditions. So May (1961) concluded that the life cycle is completed in
50 days at 27.8 C (average), similar to our result in swine meat. However pupal
period spent 9.10 days and 31 larval periods. The differences with our results
could be in the inclusion of prepupa install time in pupal period. Tomberlin et al
(2005) obtained similar results in pig liver, where Hermetia larvae reared at 27
C needed over 40 days to complete its cycle. Goff & Lord (1994) observed in a
forensic case that H. illucens takes approximately 50 days in complete the life
cycle at 27.8 C. Other references, as Tingle (1975) or Booth & Shepard (1984),
provide less information about the time of stages and differ from results present
here.
In general the length and weight of larvae breeding at 25C show a slower
growth than higher temperatures. A relation between weight and length of larvae
was observed, as other authors have found in other Diptera (Boatright &
Tomberlin, 2010; Clark et. al., 2006). In prepupa install the larvae leave the
- 92 -
trophic medium and migrate to find a quite place to pupa. In this phase, the size
of individuals usually decreased (Boatright & Tomberlin, 2010), as confirmed
our results to BSF. In the case of pupa, the weight shows the same trend, being
the pupae lighter at 25C than at higher temperatures. However in swine meat
this behaviour changed and the lighter pupae were obtained at 30C.
The number of degree-days necessary to complete development can vary
with available resources (Trudgill et al., 2005). Insects developing on high
quality resources require fewer degree-days to develop than those on lower
quality resources (Amalraj et al., 2005; De Haas et al., 2006). In this study, H.
illucens need more ADDs to complete the life cycle in swine meat than hen feed.
This may be because less quantity of energy necessary for the formation of the
adult with is accumulated with swine meat. This happens with other Diptera,
such has be seen that the larvae of Calliphora vicina and Lucilia sericata
(Calliphoridae) grow significantly faster on lung or heart tissue than on pig
organs or than the cow organs (Kaneshrajah & Turner, 2004; Clark et al., 2006;
Tomberlin et al., 2009). Hermetia illucens uses a variety of decaying organic
matter in the development of their larvae, from dung and crop residues to animal
tissues (Newton et al., 2005a; Hem et al., 2008; Myers et al., 2008). It is seen
that the larvae of this species can be adapted to a variety of foods, however larval
growth depends on the quality of the diet as well as the temperature. Moreover,
the minimum thresholds calculated varied in base to the substrate used to feed to
larvae. In hen feed was 3.9 and in swine meat was 7.3, this last more similar to
the value of 10, used by other authors than have published ADDs for H. illucens
(see in Oliveira-Costa, 2011). Probably, this is the reason so the ADD of life
cycle obtained in this study would be higher than obtained by May (1961) or
Tingle (1975) (890 and 733.9 respectively).
- 93 -
Captulo II
Regarding to the sex ratio, in the first 10 pupae analysed in each replicate,
over 50% of the emerged adults were males. These BSF females need more time
to feed and consequently weigh more than males, though our results show similar
weights in both sexes (perhaps number of sample was insufficient). The adults do
not feed, and they depend exclusively on the accumulated reserves during the
larval stage. For this reason is very important for females achieve a healthy
weight, because it will determine its reproductive fitness (Tomberlin et al.,
2009).
In terms of mortality, it increments when temperature does it. In both diets

was obtained highest mortality at 35 C, this is expected since increasing the
temperature increases the metabolism and growth by preventing the
accumulation of energy necessary for the formation and emergence adult
(Tomberlin et al., 2009). Also mortality was great in swine meat than hen feed.
We have seen that the hen feed is an ideal diet for growing H. illucens because
provides calcium necessary for larval development, larva ingests calcium and
convert it to calcium carbonate which is secreted through the hypodermis and
covers the larval integument (Johannsen, 1922). Also the calorie of pupae content
accumulated vital energy source for adults, this provided energy that result in
increased longevity of the adults (Tomberlin et al., 2002).
These results contribute to the use of the Black Soldier Fly as forensic
indicator and will allow estimate the PMI in numerous forensic cases (Lord et al.,
1994; Oliveira-Costa, 2003; Pujol-Luz et al., 2008; Martnez-Snchez et al.,
2011), demonstrating the great otential of this species in the forensic studies,
especially for calculating intervals over 15 days when most of specimens of
Calliphoridae, Sarcophagidae or Muscidae fully developed, leaving the corpse
(Ferrari et al., 2009). For this reason an isomorphic diagram and ADD help to
resolve new forensic cases. However the substrate is a fundamental factor in this
- 94 -
species, mainly to applying ADD. Therefore, it is necessary to more detailed

information about development rate to other diets and temperatures, and the
minimum threshold of development in H. illucens, which will provide key
background for future cases in forensic investigations and also applied studies of
mass-rearing and production of this species.
- 95 -
5. BIBLIOGRAPHY
AMALRAJ, D. D.; SIVAGNANAME, N. & DAS, P. K. 2005. Effect of food on

immature development, consumption rate, and relative growth rate of
Toxorhynchites splendens (Diptera: Culicidae), a predator of container
breeding mosquitoes. Mem. Inst. Oswaldo Cruz. 100:893-902.
BOATRIGHT, S. A. & TOMBERLIN, J. K. 2010. Effects of temperature and

tissue type on the development of Cochliomyia macellaria (Diptera:
Calliphoridae). J. Med. Entomol. 47:917923.
BOOTH, D. C. & SHEPPARD, D. C. 1984. Oviposition of the black soldier fly,

CATTS, E. P. & HASKELL, N. H., editors. 1990. Entomology and death: a

procedural guide. Clemsom, SC: Joyces Print Shop.
CENTENO, N. 2002. La sinantropa de Calliphoridae (Insecta: Diptera) en

Hudson, Argentina. Resmenes V Congreso Argentino de Entomologa,
Buenos Aires, p. 433.
CLARK, K.; EVANS, L. & WALL, R. 2006. Growth rates of the blowy,
Lucilia sericata, on different body tissue. Forensic Sci. Int. 156:145-149.
DE HAAS, E. M.; WAGNER, C.; KOELMANS, A. A.; KRAAK, M. H. S. &

ADMIRAAL, W. 2006. Habitat selection by chironomid larvae: fast
growth requires fast food. J. Anim. Ecol. 75:148-155.
DUNN, L. H. 1916. Hermetia illucens breeding in a human cadaver. Entomol.

News. 27:59-61.
FERRARI, A. C.; SOARES, T. C. A.; AMORIM, S. D.; THYSSEN, J. P. &

GUIMARES, A. M. 2009. Comparao dos padres de atratividade de
Hermetia illucens (Diptera, Stratiomyidae) associada a carcaas de Rattus
norvergicus enterradas e tratadas com hormnios esterides. Revista
Brasileira de Entomologia. 53(4):565569.
GOFF, M. L. 1993. Estimation of postmortem interval using arthropod

development and successional patterns. Forensic Sci. Review. 5(2):81-94.
GOFF, M. L. & LORD, W. D. 1994. Entomotoxicology: A new area for forensic

investigation. Am. J. Forensic Med. Pathol. 15:5157.
GREENBERG, B. 1991. Flies as forensic indicators. Journal of Medical

Entomology. 28:56 577.

HOWE, R. W. 1967. Temperature effects on embryonic development in insects.

Rev. Entomol. 12:15-42.
JOHANNSEN, O. A. 1922. Stratiomyiid Larv and Puparia of the North Eastern

States. Journal of the New York Entomological Society. 30(4):141-153.
KANESHRAJAH, G. & TURNER, B. 2004. Calliphora vicina larvae grow at

different rates on different body tissues. Int. J. Legal Med. 118:242-244.
LINNAEUS, C. 1758. Systema naturae per regnatri a naturae. Ed. 10. 1:1-824.
Holmiae (=Stockholm).
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Captulo II

black soldier fly Hermetia illucens (Diptera, Stratiomyidae) as a potential
measure of human postmortem interval: observations and case histories. J.
Forensic. Sci. 39:21522.

corpses in Iberian Peninsula. Forensic Science International. 206:76-78.
soldier fly Hermetia illucens (L.). New Zealand Journal Sci. 4:5565.
MYERS, H.; TOMBERLIN, J.; LAMBERT, B. & KATTES, D. 2008.

Development of Black Soldier Fly (Diptera: Stratiomyidae) Larvae Fed
Dairy Manure. Environ. Entomol. 37(1):11-15.
NEWTON, G. L.; SHEPPARD, D.C.; WATSON, D.W.; BURTLE, G. & DOVE,

R. (a) 2005. Using the black soldier fly, Hermetia illucens, as a value-
added tool for the management of swine manure. Animal and poultry
waste management center, North Carolina State University, Raleigh, NC.
17p.


Vestgios. So Paulo, Millennium, 3 edicao. 502 p
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(Diptera, Stratiomyidae), used to estimate the postmortem interval in a

SUMNER, S. M. 2002. Rearing methods for the black soldier fly (Diptera:
Stratiomyidae). J. Med. Entomol. 39:6958.
TINGLE, F. C.; MITCHELL, E. R. & COPELAND, W.W. 1975. The soldier fly,
Hermetia illucens, in poultry houses in North Central Florida. J. Ga.
TOMBERLIN, J. K. & D. C. SHEPPARD, D. C. 2001. Lekking behavior of the

black soldier fly (Diptera: Stratiomyidae). Florida Entomologist. 84:729
730.
TOMBERLIN, J. K.; SHEPPARD, D. C. & JOYCE, J. A. 2002. Selected life-

history traits of black soldier flies (Diptera: Stratiomyidae) reared on three
artificial diets. Ann Entomol. Soc. Am. 95:37986.
TOMBERLIN, J. K.; SHEPPARD, D.C. & JOYCE, J. A. 2005. Black soldier

flies (Diptera: Stratiomyidae) colonization of pig carrion in south Georgia.
J. Forensic Sci. 50:1523.
TOMBERLIN, J. K.; ADLER, H. P. & MYERS, M. H. 2009. Development of

the Black Soldier Fly (Diptera: Stratiomyidae) in Relation to Temperature.
Environ. Entomol. 38(3):930-934.
TRUDGILL, D. L.; HONEK, A.; LI, D. & VAN STRAALEN, N. M. 2005.

Thermal time-concepts and utility. Ann. Appl. Biol. 146:1-14.
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Captulo II
USTUNER, T.; HASBENLI, A. & ROZKO, R. 2003. The first record of

Hermetia illucens (Linnaeus, 1758) (Diptera, Stratiomyidae) from the
Near East. Stud. Dipter. 10:181185.
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Captulo III
Effect of larval diet on adults of Hermetia illucens
The effects of larval diet on adult life-history traits of

the Black Soldier Fly, Hermetia illucens (L.) (Diptera,
Stratiomyidae).
Abstract
Larvae of Hermetia illucens feed on different types of decomposing

organic matter, and their development depends on the quality and quantity of
food ingested. In this study three artificial diets were analyzed, namely hen feed,
meat meal and a mixture of meat meal and hen feed. The effects of diet on
ovarian development, size, mortality, larva/pupa stages and sex ratio were
studied. Results showed more biological disadvantages rearing adults using a
meat meal diet than with the other two diets; both mortality rate and the duration
of larvae/pupae were higher, and ovarian development and size of the adults were
lower; in contrast, hen diets were the best. We conclude that food ingested by the
Black Soldier Fly larvae acts as a determining factor in both physiological and
morphological development of adults.
Gobbi, P.; Martnez-Snchez, A. & Rojo, S. 2012. The effects of larval diet on adult life-history traits
of the Black Soldier Fly, Hermetia illucens (L.) (Diptera, Stratiomyidae). European Journal of
Entomology. Submitted and accepted.
- 103 -
Captulo III
1. Introduction
Use of Hermetia illucens larvae (Linnaeus, 1758) (Diptera: Stratiomyidae)

have clear advantages for the treatment of organic waste, as they process a wide
variety of organic matter, from vegetable residues to decaying animal tissue
(Newton et al., 2005a, Hem et al., 2008, Myers et al., 2008, Martnez-Snchez et
al., 2011). This versatility has been demonstrated by numerous studies carried
out on different types of residues, such as coffee chaff, hen or swine manure,
palm oil or fish processing waste (Lard, 1989; Newton et al., 2005a; St-Hilaire
et al., 2007; Hem et al., 2008). This species, known as the Black Soldier Fly
(BSF), is able to reduce 42-56 % of the volume of organic matter, by consuming
and accumulating it as protein (40 % or more) in its body (Newton et al., 2005b).
Due to the fact that adults do not need to feed (Tomberlin et al., 2002),
depending on the quantity and quality of food supplied to larvae, variations can
take place in their development and formation, as well as in their biological cycle
(Liu et al., 2008). It is known that the quality of food affects growth and survival
rate of insects. Therefore, under optimal conditions, BSF larvae take two weeks
to reach the prepupa stage, although this period can be extended to four months if
food is limited (Furman et al., 1959).
To analyse effect of larval food quality in Diptera, adult wing size is the
most widely used method for studying geometric morphometrics. This technique
provides biological information using a few variables from the anatomical
structure and provides greater statistical power to evaluate analytical and visual
differences in biological structures (Rohlf, 1993). Geometric morphometric
methods based on wings landmarck structure are a great tools to use for the study
of variability in laboratory strains of flies, such as: the influence of larval density
or diet, discrimination of different populations, etc. (Jirakanjanakit & Dujardin,
2005; Jirakanjanakit et al., 2007). It is assumed that larger specimens produce
more eggs then, size of the ovary and basal oocyte were analyzed. Ovary size
- 104 -
indicates greater development of ovarioles, which are responsible for producing

the eggs, which in the first stage of development are called basal oocytes
(Salmon et al., 1992). The development of the ovaries and number of ovarioles
in insects is genetically determined in most species, nevertheless, the quantity of
ovarioles and their body size can vary depending on the quantity and quality of
food obtained and stored during the life cycle of individuals (Magnarelli et al.,
1982; Engelman, 1984; Salmon et al., 1992).
The main aim of this study was to determine how larval diet affects the
development of H. illucens and fecundity of females. If larval diet affects the
process of mass rearing, it could be hypothesized that larger specimens produce
females with larger ovaries that lay more eggs. Alternatively size does not affect
the ovaries and the number of eggs produced is related to the quality of food. In
order to test this hypothesis and determine new biological parameters for the
Black Soldier Fly, we established the following specific objectives: a) to
determine how larval feeding affects survival b) to analyse wing size from adults
feeding on different diets, c) to know the life cycle for various larval diets and d)
to analyze the development of ovaries and oocytes in relation to female feeding.
2. Materials and Methods
This study was carried out at the University of Alicante (Alicante

province, SE Spain). A colony of Black Soldier Fly was established in 2008 from
pupae commercially available (Insect Science Resource Company, Georgia,
USA). Emerged adults were placed in colony cages of 3 m and kept in a glass
greenhouse module under controlled conditions (255 C, 5010 % RH and
natural light). Adults were provided with water and sugar ad libitum and
approximately 7 days after emergence, a mixture of water and hen feed (500 gr
diluted in 800 ml of water) was offered for oviposition. The medium was
- 105 -
Captulo III
prepared in a small container (8.5 x 8.5 cm), with surface covered with strips of
cardboard with holes along the edge, to let females lay the eggs. Every day the
container was substituted, and the medium with eggs was transferred to a
climatic chamber (25 C, 60 % RH, 12:12) to optimize hatching. Later, hen feed
medium was supplied ad libitum to developing larvae. When larvae reach
prepupal stage, they left the rearing medium to pupate in a sand tray placed at the
bottom of the container. The pupae were filtered and transferred to the adult
cages in the greenhouse.
2.1. Experimental design
Three different diets were prepared for larval feeding: hen feed (H) (500
gr diluted in 800 ml of water), a mixture of hen feed and meat meal (H+M) (250
gr hen feed+250 gr meat meal diluted in 800 ml of water) and meat meal (M)
(500 gr diluted in 800 ml of water). Five replicates with 600 first instar larvae
(younger than 24 hours) were deposited in each diet and then they were placed in
a chamber (25 C, 60 % RH and 12:12). Once all larvae pupated, as commented
above, they were transferred to experimental adult boxes (40x40x40 cm)
containing sugar and water ad libitum. The dates of pupation and adult
emergence, sex ratio and residue weight were registered; morphometics of 30
males and 30 females, and rate of larval, pupal and adult mortality were also
calculated.
To analyze their morphometric data, length of adults was measured with a

digital caliper (0.01 mm) and wings were removed for measurement. The pair
of wings from each specimen was glued to a transparent film, noting the replica
and diet reference. They were then scanned and measured using the tps software
that is based on three different programs: tps file utility program (tpsUtil), with
which tps files were created to minimize any bias; tps digitize landmarks &
- 106 -
outlines from image files, scanner or video (tpsDig), which allowed selection of
morpho-geometrical points (landmarks) from the images (in this study 21 points
were used (Figure 1); to thereby capture the configuration of each wing and
convert those points into two-dimensional coordinates; and finally, tps relative
warps analysis (tpsRewl), the principal component of the program that processes
the coordinate matrix by calculating the centroid size (values that reflect the
measure image size). In all cases the right wing was measured, but in some cases
where this was impossible the left one was used.
To know the effect of larval diet on the ovarian development of females

and size of individuals, females were analyzed at day 1 (90% emerged adults),
day 5, day 10, day 15 and day 20. Ten females were individualized and frozen in
each replica for 72 hours. Then, their abdomens were opened and ovaries stained
with blue toulidina and orange G (0.03 gr in 6 ml of distilled H2O; one minute).
Maximum width (MWO) and length (MLO) of the ovary and maximum width
(MWob) and length (MLob) of the largest basal oocyte were measured with a
micrometre (magnification 0.63 and 6, respectively). The length of females was
measured with a digital caliper (0.01 mm) and wings were extracted also to
undertaking morphometric analysis.
- 107 -
Captulo III
4 6
2 5 7
3 19
1
18
17 20
14 8
21
16
13 15
9
12 10
11
Figure 1. Hermetia illucens wing with the 21 landmarks used to perform morphometric analysis
wing.
2.2. Statistical analysis
To determine possible differences in wing size, mortality and duration of

period stages the non-parametric Kruskal-Wallis (H) test and Mann-Whitney (U)
test was used. To establish the possible relationships that may exist between wing
size and measurements of MWO, MLO, MWob and MLob, Pearson correlation
analysis was applied, and the non-parametric Mann-Whitney (U) test was used to
determine possible differences between MWO, MLO, MWob and MLob for
different diets. The program used was SigmaStat (v3.5 for Windows) and values
of p greater than 0.05 were discarded.
- 108 -
3. Results
3.1. Mortality, duration of stages and sex-ratio
Larvae were maintained under the same conditions for the three types of
diet, so the only parameters influencing larval/pupal mortality were the quantity
and quality of food ingested. Quantitative analysis of the remains of the medium
for each type of diet demonstrated that in meat meal larvae ingested an average
of 167.667.80 gr (dry weight), in hen feed this was 354.827.78 gr (dry weight)
and for the mixture the value was intermediate, 290.634.79 gr (dry weight).
Maximum larval/pupal mortality was observed in the meat meal diet, where
values reached 603 % and 802.66 %, respectively. In contrast, using hen feed,
survival was the highest with 73% of mortality rate in larvae and 10.6 % at the
pupal stage (Figure 2). In all cases the sex ratio showed more females than males,
but no significant differences were found for any of the three diets (Table 1).
H M+H M
100
*
80
Percentage (%)
*
60
40 *
20
0
Larvae mortality Pupae mortality Dry residue consumed
Figure 2. Larvae and pupae mortality (SD) and total dry residue consumed (SD) of Hermetia
illucens in hen feed (H), meat meal + hen feed (M+H) and meat meal (M) diets (*p <0.05
significant differences).
- 109 -
Captulo III
Table 1. Average (SD) adult size (males and females) and sex ratio in base to larval diets (H:
hen feed, M+H: hen feed + meat meal and M: meat meal).
Female Lenght Sex Ratio

Diets Replica Male Lenght (mm)
(mm) % Female % Male
A 15.571.02 15.661.20 62 38
B 16.081.01 16.221.16 55 45
H C 15.781.17 15.951.32 58 42
D 15.611.20 15.891.29 56 44
E 15.470.95 15.771.05 56 44
A 15.891.03 16.270.88 55 45
B 15.641.19 16.830.75 55 45
M+H C 16.021.08 16.101.31 60 40
D 15.971.11 16.080.90 58 42
E 16.011.08 16.461.20 54 46
A 8.060.87 9.450.63 58 42
B 8.340.86 9.260.74 54 46
M C 8.510.86 9.190.77 53 47
D 8.320.92 9.791.96 63 38
E 8.240.86 9.380.69 58 42
The duration of larval and pupal stages in the three treatments showed
significant differences (larva: H=12.77, p<0.005 and pupa: H=12.23, p<0.005)
(Figure 3). The larval period was similar in hen diets (H: 150.55 days and
M+H: 191 days) and was approximately 15 days shorter than in the meat meal
diet (M: 331.09 days). The pupal period showed less variation than the larvae,
160 days for hen feed and 160.45 days for hen+meat meal feed, with the
maximum for meat meal, 190.55 days. It was noted that for both hen diets (H
and M+H), it took significantly less time (30.60.55 days and 35.21.46 days,
respectively) to complete the life cycle than for meat meal, which took almost
twice as long (52.41.64 days) (H=12.68, p<0.005) (Figure 3).
- 110 -
H M+H M
60
*
50
40
*
Days
30
20 *
10
0
Larval stage duration Pupal stage duration Total duration
Figure 3. Larval, pupal and total period (medianSD) of the life cycle of Hermetia illucens in
hen feed (H), meat meal+hen feed (M+H) and meat meal (M) diets (*p<0.05 significant
differences).
3.2. Adult size and ovarian development
Wing size was used for morphometric analysis, as it is a good indicator of

the body size of Black Soldier Fly adults (r=0.99; p=0.0001). Wing size was
studied separately according to sex of individuals because significant differences
between males and females were found for the three diets (diet H: U=9740,
p<0.05; diet M+H: U=8814.5, p 0.001; M: U=1458, p 0.001). In all cases, the
wing size of females was larger than that of males (Figure 4). There were
variations in wing size in males (H=233.40, p0.001) and females (H=200.87,
p0.001) for each type of treatment. Results showed significant differences
between wing sizes for the different diets, except among males fed with both hen
diets, where the differences were not significant (Figure 4). Anyway, the hen
feed mixed with meat meal diet was the best medium to obtain large females, and
specimens developed using the single meat meal were smaller-sized when
compared to individuals fed with hen diets (Figure 4; see also adult size in Table
1).
- 111 -
Captulo III
2000
1800 * *
1600
1400
Centroid size
1200
1000 * *
800
600
400
200
0
H M+H M H M+H M
Female Male
Diets
Figure 4. Box plot showing the relative size of the centroid of females and males wings in the
three treatments (-- : Mean, : SE, T : SD) (*p<0.05 significant differences).
The mixed diet (M+H) had females with larger ovaries and basal oocytes
than females fed on hen feed or meat meal on all of the days sampled, except on
the first day when oocytes in hen diet were larger (Table 2). The maximum ovary
area was observed from the fifth day in hen diets, and the maximum development
of basal oocytes took place from the fifth day too in both diets (except tenth day
in hence diet) (Figure 5 A-B). In females obtained from meat meal diet, only
ovaries and oocytes were slightly developed on the first day after emergence. In
Figure 6 it was observed that in the mixed diet the specimens were larger, but
sometimes when females with similar size and breeding in different diets were
compared, females that emerged from hen+meat meal showed similar or larger
basal oocytes (day 10) than those on hen feed diet. In contrast, the females
developed in meat meal showed the smallest ovaries and basal oocytes only on
- 112 -
the first day sampled, due to very high mortality on the other days and there were
no females (MLO 54.114.31; MWO 14.551.42, MLob 14.430.39, MWob
14.430.39). When performing Pearson correlation analysis, a positive
relationship between adult size, wing size and ovary and basal oocyte size was
observed for all the treatments (Table 3; Figure 6). The highest coefficient values
were observed in the relation between wing size and length and not the width of
ovary and basal oocytes.
- 113 -
- 114 -
Captulo III
Table 2. Ovary (length MLO and width MWO) and basal oocyte development (meanSD) of Hermetia illucens in hen feed (H) and meat
meal+hen feed (M+H) (*p0.05) [females breeding in meat meal alone develop ovaries in the first day (see text)]
Sampling
1 5 10 15 20
days
Diets H M+H H M+H H M+H H M+H H M+H
66.14 83.08 99.44 105.34 97.6 104.72 97.64 105.24 98.12 105.22
MLO
7.90* 5.70* 7.65* 5.82* 7.18* 5.69* 6.11* 4.87* 7.18* 5.26*
19.824. 24.5 24.90 22.78 24.9 23.18 25.38 23.04 25.14

MWO 15 3.58*
79* 1.91 2.06 2.36* 2.14* 2.33* 1.83* 2.73* 1.89*
26.412. 98.08 105.18 79.16 105.18 98.02 105.22 98.7 105.82

MLob 29.92 9*
07* 6.95* 6.44* 8.18* 6.17* 6.17* 5.18* 8.16* 5.47*
24.885. 23.06 24.12 23.5 24.5 23.76 23.96 23.54 24.6

MWob 29.92 9*
46* 2.22* 1.82* 2.01* 2.13* 2.14* 2.14 2.10* 1.73*
H M+H M
10
9
A
Area of the ovary (mm)
8
7
6
5
4
3
2
1
0
1 2 3 4 5
Days after emergence
H M+H M
0.16
Area of basal oocyte (mm)
0.14
B
0.12
0.1
0.08
0.06
0.04
0.02
0
1 2 3 4 5
Days after emergence
Figure 5. Ovary (A) and basal oocyte (B) area of Hermetia illucens female in three different
diets (H: hen feed, M+H: meat meal+hen feed and M: meat meal).
- 115 -
Captulo III
MLO-H-Day 1 MLO-M+H-Day 1 MLob -H-Day 1 MLob-M+H-Day 1
10 1.2
8 1
0.8
6 0.6
4 0.4
2 0.2
0 0
1300 1500 1700 1900 1300 1500 1700 1900
MLO-H-Day 5 MLO-M+H-Day 5 MLob-H-Day 5 MLob-M+H-Day 5

10 1.2
8 1
6 0.8
0.6
4 0.4
2 0.2
0 0
1300 1500 1700 1900 1300 1500 1700 1900
SIZE OF THE OVARY (mm)

10 1.2
8 1
6 0.8
4 0.6
0.4
2 0.2
0 0
1300 1500 1700 1900 1300 1500 1700 1900

10 1.2
8 1
6 0.8
4 0.6
0.4
2 0.2
0 0
1300 1500 1700 1900 1300 1500 1700 1900

10 1.2
8 1
6 0.8
4 0.6
0.4
2 0.2
0 0
1300 1500 1700 1900 1300 1500 1700 1900
CENTROID SIZE
Figure 6. Relationship between wing size and MLO and MLob of the females during
experiment in hen feed (H) and meat meal + hen feed (M+H). No enough females were obtained
in diet meat meal (M) for carry on this analysis.
- 116 -
Table 3. Correlation coefficient (r) of linear equation between wing size and female size with
the maximum length (MLO), width of the ovary (MWO), the maximum length (MLob) and
width of the basal oocyto (MWob) in the three diets (*p0.001).
Centroid Size Female Size
Diets Day MLO MWO MLob MWob MLO MWO MLob MWob
1 0.981* 0.845* 0.948* 0.948* 0.823* 0.824* 0.799* 0.799*
5 0.874* 0.801* 0.878* 0.768* 0.821* 0.737* 0.796* 0.749*
H 10 0.978* 0.933* 0.951* 0.822* 0.832* 0.858* 0.855* 0.801*
15 0.930* 0.857* 0.925* 0.789* 0.761* 0.777* 0.827* 0.739*
20 0.983* 0.920* 0.919* 0.889* 0.848* 0.757* 0.752* 0.749*
1 0.920* 0.836* 0.981* 0.981* 0.887* 0.845* 0.732* 0.732*
5 0.982* 0.880* 0.930* 0.883* 0.916* 0.928* 0.743* 0.738*
H+M 10 0.996* 0.913* 0.962* 0.879* 0.928* 0.895* 0.747* 0.721*
15 0.996* 0.952* 0.981* 0.878* 0.943* 0.935* 0.787* 0.754*
20 0.878* 0.776* 0.925* 0.693* 0.823* 0.889* 0.784* 0.720*
M 1 0.995* 0.957* 0.998* 0.998* 0.921* 0.916* 0.846* 0.846*
- 117 -
Captulo III
4. Discussion
The larvae of the Black Soldier Fly feed on a wide variety of organic
substrates derived from plants and animals, which results in waste reduction and
transformation of these organic materials (Diener et al., 2009). The feeding phase
of the species occurs only in the larval stage, because the great fat storage
provided by the larvae appears to reduce or eliminate the need for adult feeding
(Sheppard et al., 2002). For this reason the quality of food offered to the larvae is
extremely important because the energy they store will have an important role in
the formation and subsequent development of the adult. According to Parra
(1990), during the larval stage the insects tend to choose appropriate food in
balanced proportions, so that its use promotes growth and development, giving
rise to reproductively competitive adults. In this study we found that females
obtained from larvae fed exclusively on meat meal have high mortality, long
developmental time, low wing size, and less ovarian development than those fed
with hen feed diets. This may be because the nutritional value of meat meal and
the quantity ingested by the larvae is so low that larvae spend more time feeding
in the medium, but adults do not accumulate the energy required for normal
development and reproduction. Many factors, such as body size (Livdahl, 1982;
Carpenter, 1983; Briegel, 1990a; Broadie & Bradshaw, 1991; Akoh et al., 1992;
Bradshaw & Holzapfel, 1992; Clements, 1992) and the number and size of the
ovaries of insects (Hawley, 1988; Clements, 1992) are determined by the
conditions in which the larvae develop, and this strongly affects population
growth. Numerous studies using mosquitoes have shown a positive relationship
between wing size (or other measurements of body size) and fecundity (Livdahl
& Sugihara, 1984; Packer & Corbet, 1989; Briegel, 1990a-b; Reeves, 1990;
Bradshaw & Holzapfel, 1992; Clements, 1992; Renshaw et al., 1994). Our data
show that females with larger wing size correspond to large values for body size
and are more fertile than females with smaller wing size and small body size.
- 118 -
This result is observed for hen diets, and concretely in the mixed diet where hen
feed, based on vegetable protein mainly, and meat meal with animal protein
provides nutrients to produce the largest females with larger ovaries and basal
oocytes. This is expected because lower body size reduces the abdominal cavity,
minimizing the maximum space required for ovarian development (Honek,
1993). As a conclusion, a reduction in the size of females produced a significant
effect on population dynamics (Salmon et al., 1992). The larger size of ovaries
and eggs was observed from day five to emergence in hen diets, but eggs did not
develop until the fifteenth day in the case of mixed diet.
There are many factors that limit the body size of insects, therefore it is
very important to research the history of their life (Blanckenhorn, 2000; Gotthard
et al., 2007; Pastor et al., 2011). Studies in Muscidae and other insects have
shown that a decrease in nutritional quantity and quality during the larval stage
reduces the size of adults (Black & Krafsur, 1987; Honek, 1993). Wing length is
often used as an indicator for body size in many insects. In our study we found
that BSF larvae fed on single meat meal only gave rise to adults with
significantly reduced wing size (small body size) compared to those fed with hen
feed and meat meal+hen feed; in all cases the females were significantly larger
than males.
The morphology of insects, such as wing size may be influenced by

numerous genetic and environmental variables; these variations provide relevant
information on many aspects of insect biology, mortality, fertility and sex ratio.
In this study there is a higher mortality rate in the diet of single meat meal, as
well as longer duration of larval and pupal stages. This is expected because a
decrease in the nutritional quality of the larvae significantly increases mortality
rate in the preimaginal stages (Shepherd et al., 1994). According to Roper et al.
(1996), the increase in larval development of Chrysomya megacephala (Diptera,
Calliphoridae) found in meat meal diet may be due to a delay of individuals in
- 119 -
Captulo III
assimilating the nutrients needed to obtain the indispensable minimum weight for
pupation.
Some morphological and biological traits of insects, such as fertility,

mortality and wing size can be affected drastically by the quantity and quality of
the food stored during the juvenile stages (Magnarelli & Anderson, 1979). This
factor and others play an important role in research on the natural history of BSF
because information about them is limited. For this reason it is important to know
the effectiveness of different artificial diets and their micronutrient deficiencies.
These factors may affect the production of body mass and size of individuals,
minimizing and/or eliminating the production of eggs necessary for the
continuity of the species under laboratory conditions. However, other factors
such as larval density, adult density and environmental conditions (light,
temperature etc.) are determining factors that must be studied in depth to improve
mass-rearing and applied use of BSF. Our results show that there are important
differences on larval developmental time, mortality and ovarian development in
relation to larval feeding substrate.
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suas implicaes no manejo de pragas. So Paulo: Manole. p.9-57.
PASTOR, B; IKOV, H.; KOZNEK, M.; MARTNEZ-SNCHEZ, A.;

TAK, P. & ROJO, S. 2011. Effect of the size of the pupae, adult diet,
oviposition substrate and adult population density on egg production in
Musca domestica (Diptera: Muscidae). Eur. J. Entomol. 108:587596.
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Arboviruses in California, 1943-1987. California Mosquito and Vector
Control Association, Sacramento, CA, 508 pp.
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reproductive success in the mosquito Aedes cantans. Medical and
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mosquito wings. In MARCUS, L. F.; BELLO, E. & GARCA-
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of Drosophila melanogaster life history to differences in larval density.
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SUMNER, S. M. 2002. Rearing methods for the black soldier fly
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Captulo III
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Captulo IV
Mass rearing of Hermetia illucens
Mass rearing of Hermetia illucens (Diptera:

Stratiomyidae): identifying bottlenecks in egg
production.
Abstract
Hermetia illucens or the Black Soldier Fly (BSF) is an insect of great

economic importance, due to its potential at larval stage for degrading a wide
variety of organic by-products and wastes. The transformation of organic matter
results in a highly nutritious product for feeding a wide variety of animals. This
paper provides information about some of the main bottlenecks related to mass
rearing of this species on an industrial scale. Two experiments were conducted to
explore biological parameters related to mass production of eggs: 1)
Density/housing colony experiment with four treatments, treatment A (colony
box 40x40x40 cm with 1000 adults), treatment B (40x40x40 cm box colony with
2000 adults), treatment C (80x60x80 cm box with 1000 adults) and treatment D
(80x60x80 cm box with 2000 adults) and 2) Mass rearing experiment with four
cases, treatment 1 (colony box with no periodic introduction of pupae), treatment
2 (colony box with fortnightly introduction of Hermetia pupae) treatment 3
(colony box with weekly introduction of pupae) and treatment 4 (colony box
with a weekly introduction of double the quantity of pupae in treatment 3). In
both cases it was observed that biotic and abiotic conditions, such as solar
radiation, temperature, humidity, density of adults and box colony size, are
important for optimal development of BSF.
Gobbi, P.; Martnez-Snchez, A. & Rojo, S. 2012. Mass rearing of Hermetia illucens (Diptera:
Stratiomyidae): identifying bottlenecks in egg production. Entomologia Experimentalis et Applicata.
Submitted.
- 129 -
Captulo IV
1. Introduction
Over 50 % of waste production belongs to organic types (Ojeda-Benitez et

al., 2003, Henry et al., 2006; Sharholy et al., 2006) and therefore can be
processed by specific techniques such as composting and other live technologies.
In most cases, organic by-products can be biologically transformed under
controlled conditions. Traditionally, saprophytic organisms such as fungi,
bacteria and earthworms were commonly used for treatment of organic wastes,
which transform the by-product or waste into a usable protein product of high
value (Burns, 2005; Diener et al., 2009). However, it is also known that some
insect larvae can perform this process, with Hermetia illucens L. (Diptera,
Stratiomyidae) being one of the most promising, especially in urban, industrial
and agricultural environments. The potential of BSF is related to the biological
characteristics of its larval stage, which can develop in a variety of fresh or
decomposed organic matter (Tomberlin et al., 2002). The Black Soldier Fly can
be used to remove between 60-90% of the volume of organic matter, as some
authors have shown experimentally (Tingle et al., 1975; Sheppard et al., 1994).
Moreover, the last stage of larval development of this species has great potential
for use as animal feed for pets (eg. reptiles, birds, etc.), or for use in aquaculture
or livestock feeding (Tomberlin et al., 2009). Preliminary studies suggest that the
larvae have a nutritional balance of calcium and phosphorus and high levels of
lauric acid with excellent antimicrobial and antiviral functions. Erickson et al.
(2004) and Liu et al. (2008) determined that the Black Soldier Fly larvae could
reduce Escherichia coli bacteria from chicken manure; a similar effect was found
in Salmonella populations. However, it is not known whether these bacteria serve
as food for the larvae or are suppressed to reduce infection and potential death of
the larvae (Erickson et al., 2004; Liu et al., 2008).
- 130 -
It has been shown that adults of H. illucens can survive for some time
without feeding, as they use energy reserves stored during the larval stage for
their development, but they can increase their longevity with the presence of
water (Tomberlin et al., 2002). When biotic and abiotic conditions are suitable
for their life cycle, it has been shown that sunlight stimulation and optimum
ranges of temperature and humidity (Tomberlin & Sheppard, 2002; Zhang et al.,
2005) are necessary for the reproduction of adults and for the successful
generation of progeny.
Insects are ectothermic organisms that regulate their physiological

functions according to environmental conditions (McGavin, 2001). In nature,
environmental conditions regularly affect an insects development, so that its
growth slows down or halts in what is called diapause (Deutsch et al., 2008). It is
therefore essential to know and understand the effects of biotic and abiotic
factors in the development of the Black Soldier Fly to identify their influence on
the life history of the species (Gullan & Cranston, 2000) and to understand main
bottlenecks related to mass egg production. The BSF is considered a beneficial
insect, and is of great economic importance (Yu et al., 2009). This species is
distributed in tropical and subtropical regions around the world (James, 1935;
Kovac & Rozkosny, 1995), however there is little information on its biology in
Europe where it has been known since the decade of the 60s (James, 1935; May,
1961; Booth & Sheppard, 1984; Sheppard et al., 1994).
The main objective of this work is to increase the knowledge of BSF

reproductive biology significantly, determining the biological conditions
necessary to improve mass rearing under controlled conditions and mass egg
production.
- 131 -
Captulo IV
2. Materials and Methods
The study was conducted in a glass greenhouse located in the Scientific

Park of the University of Alicante (SE, Spain), where the temperature and
humidity were under control conditions (255 C, 5010 % RH). To undertake
experiments, pupae were obtained from a colony of H. illucens established at the
facilities of the University of Alicante in 2008, obtained from commercial larvae
(Insect Science Resource Company, Georgia, USA).
In each experiment, the pupae were placed in a colony box (3m3),

consisting of a metal structure and covered with a white mesh (Figure 1).
Emerged adults were provided with water and sugar ad libitum. A proportional
mix of water and hen feed was placed in a recipient covered with cardboard
strips (Gobbi et al., submitted). The eggs were transferred to another recipient
with a mix of water and hen feed, and deposited in a growth chamber (25 C, 60
% RH, 12:12 L:D) to optimise the emergence of larvae. The medium with larvae
was situated in a container with a thin layer of sand in the bottom for pupation
until pupae formed.
- 132 -
Figure 1. Hermetia illucens adult mating boxes (length 220 cm, 140 cm width and 110 cm
height).
2.1. Adult density experiment
Colony boxes of two different sizes were used to undertake four different
treatments, each with five replicates. All experiments were carried out in the
greenhouse under controlled conditions (255 C, 6010 % RH), global solar
radiation was registered each day from 17/12/2010 to 13/01/2011. The first
treatment (A) consisted of a colony box of 40x40x40 cm (small cages) with 2000
adults of Black Soldier Fly (32 dm per adult). In the second treatment (B) 1000
adults were introduced in a small cage (64 dm per adult). In the third treatment
(C) 2000 adults were introduced in a colony box of 80x60x80 cm (medium
cages) (192 dm per adult) and in the final treatment (D), in the same type of
cage, 1000 adults were introduced (384 dm/adult). In each treatment the space
- 133 -
Captulo IV
for flies increased, from 32 dm to 384 dm, by increasing the space by x2, x6 or
x12, in each new treatment.
2.2. Mass rearing experiment

Four colony boxes of about 3 m (Figure 1) with each used for four
different treatments from 18/02/2010 to 18/08/2010. In each treatment the

number of pupae initially introduced was 5.780.63 kg. In the first treatment
[T0], no pupae were introduced periodically in the box. In the second treatment
[TF], 1.480.33 kg of pupae were introduced in the colony box fortnightly. In the
next treatment [TW], together with the initial quantity of pupae, a similar
quantity was introduced weekly (an average of 1.880.46 kg of pupae), and in
the last treatment [TDW], weekly introduction of double the quantity of pupae
(3.011.05 kg). We tried to introduce a similar initial amount in all treatments;
however, in some cases this was not possible. Due to the experiment taking place
on different dates, temperature, humidity and global solar radiation were
registered for each day of treatment (Figure 2).
2.3. Data analysis and statistic
Sex ratio and number of eggs were recorded in each experiment. In the
case of the mass rearing experiment, dead adults were removed every two weeks,
and the mortality rate was calculated as the initial number of pupae minus the
dead adults collected in two weeks, dividing the initial adult concentration in this
period. Sex ratio was performed dividing the females/males number by the total
number of individuals. In order to determine the average number of eggs, 30 egg
masses were counted , and then this average was used to count eggs per clutch (1
mass = 541.842.15 eggs). Moreover, 1 kg of pupae was estimated as
14102.562790.29 pupae (1 pupa = 0.070.02).
- 134 -
In both experiments the results were analysed under the assumption of

non-normal data, as they failed the Kolmogorov-Smirnov normality test. To
analyse the egg production from different treatments in both experiments and the
mortality rate for different treatments in the mass rearing experiment, we used
the non-parametric Kruskal-Wallis (H) test. For the density experiment to
calculate solar radiation and egg masses number correlation for the different
treatments the Pearson Correlation analysis was used. In both cases possible
differences in the sex ratio were determined with the non-parametric Mann-
Whitney (U) test. In all cases the statistical program chosen was SigmaStat
(version 3.5 for Windows), discriminating values of p greater than 0.05.
- 135 -
Captulo IV
Maximum Global Radiation

1800
1400
W/m
1000
600
200
Average Humidity (RH)

90
75
RH (%)
60
45
30
Average Temperature
35
30
T (C)
25
20
15
TF
T0
TW
TDW
Date of treatments
Figure 2. Maximum radiation, average relative humidity and average temperature recorded in
the treatments with Hermetia illucens. [T0: without introduction of pupae (22/02//2010-
14/04/2010), TF: fortnightly introduction of pupae (18/02/2010-04/04/2010), TW: weekly
introduction of pupae (08/03/2010-12/08/2010) and weekly double introduction of pupae
(15/04/2010-18/08/2010)].
- 136 -
3. Results
3.1. Effect of density on the production of eggs
Temperature and humidity were constant during the experiment, but

global radiation was not, which fluctuated sharply daily. Figure 3 shows how
maximum daily radiation affects BSF oviposition. When sunlight intensity
increased egg numbers incremented in all cases, whereas when solar radiation
decreased by 600-700 W/m2 fewer egg numbers were obtained. However, from
approximately day 15 of the experiment, all treatments showed a decrease in
daily oviposition regardless of subsequent radiation increases. In all treatments
the correlation between egg mass numbers and radiation was positive, at least in
the period of maximum egg production, i.e. in the first 15 days (Table 1;
treatment A: r=0.69, p=0.004, treatment B: r=0.53, p = 0.04; treatment C:
r=0.53, p<0.04 and treatment D: r=0.58, p=0.02).
Table 1. Value of correlation between daily egg production and radiation in the first 15 days of
Adult Density Experiment and total eggs and average production of eggs per female in each
treatment of this experiment (*Sex-ratio considered as 1:1; **p<0.05).
Adults Space by Adult Total Eggs

Size Colony Box N Eggs/Female*
(cm) Density (dm) Production
2000 32 567804270 14.192.39

40x40x40
1000 64 693503498 34.673.91**
40x40x40
2000 192 962236815** 24.53.81
80x60x80
1000 384 580805642 29.046.31
80x60x80
- 137 -
Captulo IV
Small cage 2000 adults Maximum radiation

20 1000
16 800
12 600
8 400
4 200
0 0
1 4 7 10 13 16 19 22 25 28
Small cage 1000 adults
20 1000
AVERAGE NUMBER OF EGG MASS
16 800
MAXIMUM RADIATION (W/m)

12 600
8 400
4 200
0 0
1 4 7 10 13 16 19 22 25 28
Median cage 2000 adults
20 1000
16 800
12 600
8 400
4 200
0 0
1 4 7 10 13 16 19 22 25 28
Median cage 1000 adults

20 1000
16 800
12 600
8 400
4 200
0 0
1 4 7 10 13 16 19 22 25 28
Days
Figure 3. Average number of egg production in relation to maximum radiation (W/m) in each
treatment during Adult Density Experiment.
- 138 -
Egg production increased as density decreased and the space per fly
increased in the cages. The highest egg production was observed in the medium
cage with 2000 adults (192 dm3/fly) (H=15.06, p=0.002), while the lowest egg
number was obtained in the small colony box with 2000 adults of the Black
Soldier Fly (32 dm3/fly) (Figure 4). When the density was very low and space per
fly was maximum, medium cages with 1000 adults, production was similar to
that obtained in small cages (Figure 5). However, Table 1 shows that higher egg
production per female occurs significantly in the treatment where small cages
and 1000 flies were disposed (H=55.40, p=0.001). Regarding the adults
introduced in boxes, after they died sex ratio was studied and the proportion
between males and females was similar in all treatments, i.e. 1:1 (p<0.05).
- 139 -
Captulo IV
Small cage 2000 adults Small cage 1000 adults

200 200
3
32 dm /fly 64 dm3/fly
160 160
120 120
80 80
AVERAGE DAYLY EGGS ACCUMULATED
40 40
0 0
1 4 7 10 13 16 19 22 25 28 1 4 7 10 13 16 19 22 25 28
Day Day
Median cage 2000 adults Median cage 1000 adults
200 200
192 dm /fly3 384 dm3/fly
160 160
120 120
80 80
40 40
0 0
1 4 7 10 13 16 19 22 25 28 1 4 7 10 13 16 19 22 25 28
Day Day
Figure 4. Average daily eggs accumulated ( SD) in every treatment during the adult density
experiment (small cage: 40x40x40 cm and median cage: 80x60x80 cm) of Hermetia illucens.
- 140 -
1000 adults 2000 adults

120000
100000
Total egg number
80000
60000
40000
Small colony box Median colony box
Figure 5. Total number of egg in small and median cages colonies at different adults densities.
Different letters indicate significant differences (p0.05).
3.2. Experiment mass rearing with different protocols
Comparing egg production with the number of live adults at intervals of

15 days according to different treatments, it is observed that in treatment T0 the
difference between egg numbers collected and the quantity of adults is much
higher. We interpret that egg production is greater than in the other treatments
where these quantities do not differ much (Figure 6). In all treatments the highest
egg production of BSF females was between days 1-30 of the experiment. After
this point, even though the number of adults alive remained constant or increased
in all treatment, egg production decreased generally, except for T0 treatment.
- 141 -
Captulo IV
Treatment T0 Treatment TF
250 1000 250 1000
200 800 200 800
150 600 150 600

NUMBER OF ACUMULATED LIVE ADULTS
100 400 100 400
TOTAL N OF EGGS ( in thousands)

50 200 50 200
0 0 0 0
1-15 15-30 30-45 1-15 15-30 30-45
Days Days
Treatment TW Treatment TDW

250 1000 250 1000
200 800 200 800
150 600 150 600
100 400
100 400
50 200
50 200
0 0
1-15
15-30
30-45
45-60
60-75
75-90
90-105
105-120
120-135
0 0
1-15
15-30
30-45
45-60
60-75
75-90
Days Days
Figure 6. Eggs production at intervals of 15 days during of the experiment of mass rearing.
(Treatments T0: no pupae introduced, TF: pupae introduced fortnightly, TW: pupae introduced,
weekly and TDW: double amount of pupae than in TW introduced weekly) (Black point: adults
alive, White point: eggs collected).
- 142 -
When the number of eggs had been compared in all treatments, correction
by individuals and time of treatment took place. This ratio (eggs/day/adult)
showed significant differences between some treatments (H=56.48, p0.001),
with the colony boxes without and fortnightly introduction of pupae (T0 and TF)
showing significantly higher values (Table 2). This result contrasts with the
number of adults and the total number of eggs, due to the fact that in treatment
T0 and TF there were far fewer adults than in the rest of treatments, and the
number of eggs in treatment TW was twice that of treatment T0, where the adults
introduced were more than twice those of T0. As a conclusion, increasing adult
numbers does not implicate a proportional increase in egg production.
Table 2. Total number of adults introduced, total egg production and eggs per adult per day in
each treatments (*p 0.05). (T0: no pupae introduced, TF: pupae introduced fortnightly, TW:
pupae introduced, weekly and TDW: double amount of pupae than in TW introduced weekly).
Experiment Eggs
N Total Adults N Total Eggs
Days (N/adult/day)
Treatment T0 45 49028 1358610 0.61*
Treatment TF 45 106818 1093914 0.22*
Treatment TW 135 320909 2824456 0.06
Treatment TDW 90 434564 1812600 0.05
As regards mortality rate, the values were lower than 0.5 in treatments TW
and TDW, except when the number of adults introduced was sporadically very
high, due to an increase in mortality. In both treatments, the mortality rate
follows a sine curve, increasing and decreasing according to adult age or its
incorporation in the boxes (Figure 7). This ratio was lower than 0.5 in T0 during
the first 45 days, but later mortality increased suddenly. In the TF treatment the
mortality rate was very low during the first 30 days, but then mortality increased
- 143 -
Captulo IV
to 0.6 and continued to grow exponentially. In any case significant differences

were found (H=11.33, p=0.01) among the treatments, with the mortality rate of
treatment TDW being higher than treatment T0.
1 120 1 120
Treatment T0 Treatment TF
0.8 100 100
0.8
80 80
0.6 0.6
60 60
0.4 0.4
40 40
AGGREGATE NUMBER OF ADULTS

0.2 20 0.2 20
PROPORTION OF ADULTS
0 0 0 0
1-15 15-30 30-45 45-60 1-15 15-30 30-45 45-60
Days interval Days interval
1 120 1 120
Treatment TW Treatment TDW
100 0.8 100
0.8
80 80
0.6 0.6
60 60
0.4 0.4
40 40
0.2 0.2 20
20
0 0 0 0
1-15
15-30
30-45
45-60
60-75
75-90
90-105
90-105
105-120
120-135
135-155
1-15
15-30
30-45
45-60
60-75
75-90
Days interval Days interval
Figure 7. Mortality proportion in relation to the added adults number during the different
treatments [T0: no pupae introduced, TF: pupae introduced fortnightly, TW: pupae introduced,
weekly and TDW: double amount of pupae than in TW introduced weekly]. (Bars: aggregate
number of adults; line: mortality proportion).
- 144 -
4. Discussion
Insects and other arthropods can play an important role in our human
economy, including the pharmaceutical industry and agriculture (Guzmn-
Mendoza, 2010). However these roles are related to the knowledge of the biology
and development of species (Peters & Barbosa, 1977).
The Black Soldier Fly has a high potential for use in different fields
related to humans (as food and feed, degradation of organic wastes, new sources
of bio-components, etc.). However to know the main biological parameters and
bottlenecks related to mass rearing and mass-production at industrial scale is
fundamental. In this study it was observed that daily global radiation played a
more important role in the BSFs oviposition than other abiotic parameters such
as temperature and relative humidity. Our results indicate that radiation higher
than 600 W/m is enough to increase production of eggs in H. illucens. Zhang et
al. (2010) found that with 500 W/m, mating and eggs were observed, and when
light was removed a reduction in mating and adult oviposition occured.
Incontrast, Tomberlin & Sheppard (2002) found that light intensity has a higher
influence on mating than on egg laying. In the present study oviposition and the
number of viable eggs were the only factors taken into consideration.
It is known that temperature and humidity affect the physiology,

development, longevity and oviposition of the species (Gullan & Cranston,
2000). During our experiments temperature and humidity were kept between 25
and 30 C, as they are considered optimal ranges for Black Soldier Fly breeding.
Both & Sheppard (1984) reported that mating and oviposition occurred between
ranges from 27.5 to 37.5 C and 30-90 % of relative humidity. So, in this study
these factors did not significantly influence the results.
Concerning the size of colony boxes, Tingle et al. (1975) analysed

reproductive behaviour of H. illucens adults in cages of 38x46x38 cm with bad
- 145 -
Captulo IV
space for adult mating, however they were unable to establish a culture with
multiple generations (Sheppard et al., 2002). In our study females laid high
numbers of eggs in smaller cages of 40x40x40 cm (1000 adults) and in cages of 3
m (50000 adults).
The density influence on adult insects being reared under laboratory

conditions is very important, but has rarely been investigated (Peters & Barbosa,
1977). It has been seen that size, growth rate, metabolism, and fertility behaviour
of different insect populations are affected by density (Peters & Barbosa, 1977).
In both experiments (adult density and mass rearing experiment) it was observed
that there is a density appropriate for each colony box size where H. illucens
development is optimal; if this limit (small colony box [40x40x40 cm] with 1000
adults, medium colony box [80x60x80 cm] with 1000 adults and colony box of
3m with 50000 adults) is exceeded, adult mortality increases and the egg laying
number per female per day decreases. In the first experiment the maximum
oviposition per female was recorded in small cages with 1000 adults, i.e. 0.064
m3 per fly. When this space increases 3 or 6 times or is reduced by half,
oviposition per female is lower or similar, respectively. However, we observed
that 1000 flies in small or medium cages produce similar quantities of eggs per
female. However space optimisation for mass rearing decreases if medium cages
are used. In medium cages with 2000 flies, total egg production was significantly
higher than for the rest of treatments, but lower than the double quantity with the
twice the number of flies. In the mass rearing study, treatment T0 (without
introduction of pupae) and treatment TF (fortnightly introduction of pupae) have
significantly higher fertility per female than treatments TW (weekly introduction
pupae) and TDW (weekly introduction of double pupae amount), with the last
one having a significantly higher mortality rate. In most insects, when population
density increases, offspring per female per day decreases; this has been attributed
to a reduction in fertility, as well as to an increase in mortality (Boyce, 1946).
- 146 -
Crombie (1942) showed that the fertility decrease of some beetle females was
due to competition from gravid females for oviposition sites. However, when H.
illucens breeding does not occur under optimal biological conditions, the
reproductive capacity of females decreases, resulting in the death of individuals
(Tomberlin & Sheppard, 2002; Tomberlin et al., 2002).
The Black Soldier Fly can be applied to solve some important problems
related to the accumulation of organic waste (odour removal, elimination and/or
reduction of other flies, elimination of microorganisms harmful to human health).
It can also be used as a substitute or complement in the food diet for different
animals, as well as intervening and helping to resolve cases of a judicial nature in
the calculation of the postmortem interval (PMI) (Erickson et al., 2004; Newton
et al., 2005; Hem et al., 2008; Myers et al., 2008; Diener et al., 2009; Martnez-
Snchez et al., 2011). It is for this reason that the present study solves many
unknowns regarding biological parameters that may affect their breeding and
development. However, deeper studies are therefore needed to expand artificial
rearing methods for this species.
- 147 -
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Temengor Forest Reserve, Hulu Perak, Malaysia. Malayan Nature
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2008. Black Soldier Fly (Diptera: Stratiomyidae) Larvae Reduce
Escherichia coli in Dairy Manure. Environmental Entomology. 37:1525-
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- 149 -
Captulo IV

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65.
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Oxford: Oxford University Press.
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Development of black soldier fly (Diptera: Stratiomyidae) larvae fed dairy
manure. Environmental Entomology. 37:11-15.
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DOVE, R. 2005. Using the black soldier, Hermetia illucens, as a value-
added tool for the management of swine manure.
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E. 2003. Characterization and quantification of household solid wastes in
a Mexican city. Resources, Conservation and Recycling. 39:211222.
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fecundity, and developmental rate of insects in culture. Ann. Rev. Entomb.
22:431-50.
SHARHOLY, M.; AHMAD, K.; VAISHYA, R. C. & GUPTA, R. D. 2006.

Municipal solid waste characteristics and management in Allahabad,
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- 150 -

Bioresource. Tech. 50:275-279.

SUMNER, S. M. 2002. Rearing methods for the black soldier (Diptera:
Stratiomyidae). J. Med. Entomol. 39:695-698.
TINGLE, F. C.; MITCHELL, E. R. & COPELAND, W. W. 1975. The soldier

fly, Hermetia illucens, in poultry houses in North Central Florida. J. Ga.
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Oviposition of Black Soldier Flies (Diptera: Stratiomyidae) in a Colony. J.
Entomol. Sci. 37 (4):345-352.
TOMBERLIN, J. K.; SHEPPARD, D. C. & JOYCE, J. A. 2002. Selected life-

history traits of black soldier fly (Diptera: Stratiomyidae) reared on three
artificial diets. Ann. Entomol. Soc. Am. 95:379-386.
TOMBERLIN, J. K.; ADLER, P. H. & MYERS, H. M. 2009. Development of

the Black Soldier Fly (Diptera: Stratiomyidae) in Relation to Temperature.
Environ. Entomol. 38(3):930-934.
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animal feedstuff. Chin. Bull. Entomol. 46:41-45. (in Chinese).
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Captulo IV
ZHANG, S. Q.; ZHANG, F. D.; LIU, X. M.; WANG, Y. J.; ZOU, S. W. & HE,
X. S. 2005. Determination and analysis on main harmful composition in
excrement of scale livestock and poultry feedlots. Plant Nutri. Fert. Sci.
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2442.
- 152 -
CONCLUSIONES
A continuacin se detallan los principales resultados y conclusiones de la tesis,

referidos a los objetivos planteados en la misma.
1.- Se presenta por vez primera la quetotaxia preimaginal caracterstica de

Hermetia illucens que, sin embargo, no presenta valor diagnstico para la
diferenciacin de los estadios larvarios.
2.- El tamao de la cpsula ceflica de Hermetia illucens, as como la morfologa

y el tamao de los espirculos posteriores, pueden utilizarse como indicadores de
la edad de las larvas; a medida que incrementa la edad larval aumenta el tamao
de la cpsula ceflica, de los espirculos posteriores y el nmero de aberturas
espiraculares. Estas estructuras permiten no solo datar las larvas sino tambin
establecer diferencias entre los estadios larvarios.
3.- El anlisis cuticular de los diferentes hidrocarburos de las larvas de H.

illucens siguen un patrn con respecto a la edad. La media de la abundancia de
los hidrocarburos aumenta con la edad y la abundancia de los hidrocarburos ms
destacados, correspondientes a los tiempos de retencin de 3, 8, 9 y 11 minutos
aproximadamente, pueden ser a priori utilizados en su conjunto para diferenciar
la edad de las larvas.
- 153 -
4.- La tasa de desarrollo preimaginal de Hermetia illucens, as como su tamao
en longitud y peso, est directamente relacionada con la temperatura y la dieta,
disminuyendo con el incremento de la temperatura o con dietas no ptimas,
como las crnicas frente a las granvoras. Sin embargo, el periodo de pupa
presenta unos patrones distintos. La duracin es ms o menos estable, salvo en
pupas dispuestas a 35 C que necesitaron ms tiempo para completar su
desarrollo que a temperaturas inferiores. El peso es menor a temperaturas
inferiores que superiores, sin mostrar un patrn bien diferenciado. El aumento de
la temperatura durante desarrollo preimaginal aumenta el metabolismo y la tasa
de crecimiento, sin embargo implica una mayor mortalidad.
5.- El nmero de grados-das necesarios para el desarrollo completo de Hermetia

illucens vara con la calidad del medio larvario disponible y con la temperatura.
Las larvas desarrolladas sobre pienso compuesto (recurso de alta calidad)
requirieron un menor nmero de grados-da que las desarrolladas en carne de
cerdo (recurso de menor calidad). En cuanto a la temperatura, los grados-das
necesarios para completar el desarrollo o bien un estadio determinado es mayor a
30C que a 25C o 35C, salvo en las pupas, donde es mayor a 35C.
6.- Los imagos del sexo femenino, procedentes de larvas alimentadas

exclusivamente sobre harinas crnicas poseen alta mortalidad, tiempo de
maduracin elevado, un menor tamao alar y menor desarrollo ovrico que los
alimentados con dietas a base de pienso compuesto. El desarrollo ovrico se
optimiza con una dieta combinada de pienso compuesto y harinas crnicas,
mientras que el mximo tamao y menor mortalidad se da en la dieta de pienso
compuesto.
- 154 -
7.- La radiacin global diaria, de origen natural, juega un papel muy importante
en la ovoposicin de H. illucens; valores superiores a 600 W/m2 son necesarios
para optimizar la produccin de huevos de los imagos.
8.- La densidad de imagos y el tamao de las cajas de cra influyen en su

potencial de cra y produccin masiva. Las relaciones ptimas para maximizar la
produccin de huevos fueron cajas de cra de 40x40x40 cm (con 1.000 adultos) y
cajas de cra de 3 metros cbicos (con 50.000 adultos). La optimizacin de dichas
cajas se consigue mediante el reemplazo de individuos quincenalmente, frente a
la introduccin constante de individuos en periodos semanales, quincenales o
mensuales.
- 155 -

Biología Reproductiva de Hermetia Illucens

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Biologa reproductiva y caracterizacin morfolgica

de los estadios larvarios de Hermetia illucens

Flavia Paola Gobbi

Flavia Paola Gobbi

Centro Iberoamericano de la Biodiversidad

Instituto Universitario de Investigacin, Universidad de Alicante.

Programa de Doctorado: Biodiversidad Gestin y Conservacin de las Especies

Dr. Santos Rojo Velasco Dra. Ana Isabel Martnez Snchez

En primer lugar agradezco a mis directores, el Dr. Santos Rojo y la Dra.

Me gustara dar las gracias a la empresa Flysoil S. A. ya que han

Al Ministerio de Ciencia e Innovacin por otorgarme la beca

Tambin quisiera agradecer a la empresa Agriprotein S. A. por permitirme

Tambin tengo que agradecer a los directivos, administrativos y al

Especial agradecimiento al personal de los Servicios Tcnicos de

Por ltimo, me gustara expresar mi ms profundo agradecimiento a mi

1.1. Parmetros biolgicos de Hermetia illucens......13

1.2. Importancia econmica de Hermetia illucens....16

1.3. Hermetia illucens como agente implicado en el clculo del intervalo

2. Objetivos y estructura de la tesis..........19

CAPTULO I: Estudio de la morfologa larvaria y anlisis preliminar de la

2. 1. Caracterizacin morfolgica de las larvas.....36

2. 2. Cuantificacin de hidrocarburos cuticulares..52

2.1. Experimental design.............106

2.2. Statistical analysis.............108

3.1. Mortality, duration of stages and sex-ratio...........109

3.2. Adult size and ovarian development.......111

2.1. Adult density experime.............133

2.2. Mass rearing experiment...........134

2.3. Data analysis and statistic.............134

3.1. Effect of density on the production of eggs......137

3.2. Experiment mass rearing with different protocols....141

Hermetia illucens (Linnaeus, 1758) es un dptero estratiomido (Diptera,

Se analiz la morfologa de los diferentes estadios larvarios y fases

A continuacin, se determinaron los requerimientos trmicos o suma

Tambin se analiz el efecto de tres medios de desarrollo larvario (pienso

Por ltimo, con objeto de identificar y resolver los cuellos de botella

Hermetia illucens (Linnaeus, 1758) is a dipterous stratiomido (Diptera,

The following requirements were determined thermal or heat summation

Finally, in order to identify and resolve bottlenecks related to the mass

El orden Diptera constituye uno de los principales grupos de insectos,

Los Brachycera son el grupo ms diversificado de dpteros, y a l

Hermetia illucens (Linnaeus, 1758) (Figura 1) conocida como mosca

humana se ha distribuido por todas las regiones tropicales hmedas y

Figura 1. Adulto de Hermetia illucens (de www.CritterZone.com).

Los adultos, probablemente presentan una dieta florcola en condiciones

causar miasis accidental en el ser humano (James, 1947; Caldern-Arguedas et

Los imagos son muy variados en forma y coloracin, presentando un

Los imagos presentan una pigmentacin predominantemente oscura, con

1.1. Parmetros biolgicos de Hermetia illucens

Aproximadamente cinco das despus de la emergencia del adulto puede

Tras el apareamiento las hembras depositan alrededor de 600 huevos en

Los adultos emergen aproximadamente despus de dos semanas tras la

En Hermetia illucens al igual que en la mayor parte de los insectos, la

Al igual que la temperatura, la humedad ambiental puede tener

descrito diversas estrategias durante la ovoposicin, tales como la agrupacin de

Por otro lado, algunos estudios indican que determinadas caractersticas

Otros factores como la calidad y cantidad de alimento as como tambin la

et al., 1994) permitiendo su adaptacin a diferentes tipos de hbitats y medios de

Para la cra en cautividad de Hermetia illucens, Tingle y colaboradores

1.2. Importancia econmica de Hermetia illucens

La gestin de restos orgnicos mediante insectos trasforma estos en

demostrando su utilidad como fuente de protena cruda y lpidos altamente

Otro interesante subproducto derivado de la utilizacin de larvas de H.