1
10
4435
-
https://biblioteca.senasa.gob.ar/files/original/9d836819763b56008de94293858016ea.pdf
ee1dae90ea30766f2c2e3aa087d911cd
PDF Text
Text
https://www.boletinoficial.gob.ar/#!DetalleNorma/226948/20200318
ADMINISTRACIÓN FEDERAL DE INGRESOS PÚBLICOS
Resolución General 4682/2020
RESOG-2020-4682-E-AFIP-AFIP - Procedimiento. Cómputo de plazos respecto de la materia impositiva,
aduanera y de los recursos de la seguridad social. Resolución General N° 1.983, sus modificatorias
y complementarias. Norma complementaria.
Ciudad de Buenos Aires, 17/03/2020
VISTO la Actuación SIGEA N° 10462-36-2020 del Registro de esta Administración Federal, y
CONSIDERANDO:
Que la Resolución General N° 1.983, sus modificatorias y complementarias, dispuso que durante determinados
períodos del año -atendiendo a la ferias judiciales que se establezcan cada año para el Poder Judicial de la
Nación-, no se computen los plazos previstos en los distintos procedimientos vigentes ante este Organismo,
vinculados a la aplicación, percepción y fiscalización de los tributos a su cargo.
Que en virtud de razones de salud pública, originadas en la propagación a nivel mundial, regional y local de
distintos casos de coronavirus (COVID-19), la Corte Suprema de Justicia de la Nación previó a través de la
Acordada N° 4/20, declarar inhábiles los días 16 al 31 de marzo del corriente año, ambos inclusive, para las
actuaciones judiciales ante todos los tribunales que integran el Poder Judicial de la Nación.
Que en concordancia con ello, resulta aconsejable adoptar idéntico criterio en el ámbito de esta Administración
Federal, a los fines indicados en el primer considerando de la presente.
Que han tomado la intervención que les compete la Dirección de Legislación y las Subdirecciones Generales de
Asuntos Jurídicos, Recaudación, Sistemas y Telecomunicaciones y Servicios al Contribuyente.
Que la presente se dicta en ejercicio de las facultades conferidas por los artículos 6° y 7° del Decreto N° 618 del 10
de julio de 1997, sus modificatorios y sus complementarios.
Por ello,
LA ADMINISTRADORA FEDERAL DE LA ADMINISTRACIÓN FEDERAL DE INGRESOS PÚBLICOS
RESUELVE:
ARTÍCULO 1°.- Fijar entre los días 18 al 31 de marzo de 2020, ambos inclusive, un período de feria fiscal
extraordinario con el alcance de las previsiones de la Resolución General N° 1.983, sus modificatorias y
complementarias.
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ARTÍCULO 2°.- La presente entrará en vigencia el día de su publicación en el Boletín Oficial.
ARTÍCULO 3°.- Comuníquese, dese a la Dirección Nacional de Registro Oficial para su publicación en el Boletín
Oficial y archívese. Mercedes Marco del Pont
e. 18/03/2020 N° 15564/20 v. 18/03/2020
Fecha de publicación 18/03/2020
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�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Otros organismos
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Resolución General N° 4682/2020 AFIP
Description
An account of the resource
Período de feria fiscal extraordinario
Source
A related resource from which the described resource is derived
<a href="http://servicios.infoleg.gob.ar/infolegInternet/verNorma.do?id=335646">Resolución General N° 4682/2020</a>
Language
A language of the resource
Es
Date
A point or period of time associated with an event in the lifecycle of the resource
Martes 17 de Marzo de 2020
Type
The nature or genre of the resource
Resolución
Abstract
A summary of the resource.
Se fija entre los días 18 al 31 de marzo de 2020, ambos inclusive, un período de feria fiscal extraordinario con el alcance de las previsiones de la Resolución General N° 1.983, sus modificatorias y complementarias.
Creator
An entity primarily responsible for making the resource
Administración Federal de Ingresos Públicos
-
https://biblioteca.senasa.gob.ar/files/original/71b6c01ac6330c59b347e258f9c587c9.pdf
f07f69d8b8b025c493e4793108c4b057
PDF Text
Text
https://www.boletinoficial.gob.ar/#!DetalleNorma/227420/20200403
ADMINISTRACIÓN GENERAL DE PUERTOS
Disposición 36/2020
DI-2020-36-APN-GG#AGP
Ciudad de Buenos Aires, 02/04/2020
VISTO el Expediente Nº EX-2020-19614700- -APN-MEG#AGP, la Ley N° 27.541 y el Decreto de Necesidad y
Urgencia N° 260 del 12 de marzo de 2020, y
CONSIDERANDO:
Que el PODER EJECUTIVO NACIONAL, a través del Decreto de Necesidad y Urgencia N° 260/20, amplió la
emergencia pública en materia sanitaria establecida por la Ley N° 27.541, a causa de la pandemia declarada por la
ORGANIZACIÓN MUNDIAL DE LA SALUD, en relación con el Coronavirus (COVID-19), por el plazo de UN (1) año,
a partir de su vigencia.
Que, en ese marco, el PODER EJECUTIVO NACIONAL ha adoptado diversas medidas tendientes a resguardar la
salud pública, resultando oportuno arbitrar los medios necesarios para cooperar en la implementación de cualquier
mecanismo o política y aunar esfuerzos para mitigar los efectos resultantes de la propagación de la enfermedad.
Que, en ese orden, el TRANSPORTE FLUVIAL y MARÍTIMO es una actividad indispensable para garantizar la
circulación de bienes y personas, en condiciones de continuidad y regularidad y, teniendo en consideración las
particularidades que se verifican en cada uno de los distintos sectores que prestan servicios de transporte,
corresponde abordar la problemática de dicho sector, a los efectos de colaborar con los lineamientos definidos por
la Autoridad Sanitaria.
Que, en tal sentido, y en virtud de razones de salud pública referidas en los Considerandos precedentes, el
MINISTERIO DE TRANSPORTE DE LA NACIÓN, a través del ACTA-2020-18334265-APN-SECGT#MTR, de fecha
20 de marzo de 2020, aprobó el PROTOCOLO DE APLICACIÓN NACIONAL COMITÉ DE CRISIS PREVENCIÓN
COVID-19 EN EL TRANSPORTE FLUVIAL, MARÍTIMO Y LACUSTRE, publicado en el Boletín Oficial en la misma
fecha.
Que, consecuentemente, deben arbitrarse las medidas conducentes para dictar un protocolo de actuación que, en
el ámbito del TRANSPORTE FLUVIAL y MARÍTIMO LACUSTRE, permita controlar la propagación del
CORONAVIRUS (COVID-19), en jurisdicción de esta ADMINISTARACIÓN GENERAL DE PUERTOS SOCIEDAD
DEL ESTADO.
Que la GERENCIA DE ASUNTOS JURÍDICOS tomó la intervención de su competencia.
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Que el suscripto, atento a lo normado en el Estatuto de la ADMINISTRACIÓN GENERAL DE PUERTOS
SOCIEDAD DEL ESTADO, aprobado por el Decreto N° 1456/87 y las Resoluciones Nros. RESOL-2018-137-APN-AGP#MTR, y sus modificatorias, y RESOL-2020-40-APN-MTR, se encuentra facultado para emitir la presente
Disposición.
Por ello,
EL GERENTE GENERAL DE LA ADMINISTRACIÓN GENERAL DE PUERTOS SOCIEDAD DEL ESTADO
DISPONE:
ARTÍCULO 1º.- Apruébese el “PROTOCOLO DE APLICACIÓN EN EL AMBITO DEL PUERTO BUENOS AIRES
FRENTE A LA PROPAGACION DEL CORONAVIRUS (COVID-19)” que como ANEXO
(IF-2020-19653871-APN-GG#AGP), forma parte integrante de la presente, sin perjuicio de la aplicación de medidas
que establezcan restricciones u obligaciones temporales diferentes, aprobadas por la Autoridad Sanitaria.
ARTÍCULO 2°.- La presente Disposición entrará en vigencia a partir de su publicación en el BOLETIN OFICIAL DE
LA REPÚBLICA ARGENTINA.
ARTÍCULO 3º.- Por la SUBGERENCIA DE ASISTENCIA ADMINISTRATIVA de la GERENCIA GENERAL,
comuníquese a todas las Dependencias de la ADMINISTRACIÓN GENERAL DE PUERTOS SOCIEDAD DEL
ESTADO y publíquese por UN (1) día en el BOLETÍN OFICIAL DE LA REPÚBLICA ARGENTINA. Por la
GERENCIA DE COMUNICACIÓN Y ASUNTOS INSTITUCIONALES, publíquese el Anexo de la presente
disposición en la página web de esta Sociedad del Estado. Oportunamente, archívese.- José Beni
NOTA: El/los Anexo/s que integra/n este(a) Disposición se publican en la edición web del BORA
-www.boletinoficial.gob.are. 03/04/2020 N° 16370/20 v. 03/04/2020
Fecha de publicación 03/04/2020
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�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Otros organismos
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Disposición AGP N° 0036/2020
Description
An account of the resource
Se aprueba el “Protocolo de Aplicación en el ámbito del Puerto Buenos Aires frente a la propagación del Coronavirus (COVID-19)”.
Source
A related resource from which the described resource is derived
<a href="http://servicios.infoleg.gob.ar/infolegInternet/verNorma.do?id=336057">Disposición AGP N° 0036/2020</a>
Date
A point or period of time associated with an event in the lifecycle of the resource
Jueves 2 de Abril de 2020
Language
A language of the resource
Es
Date Issued
Date of formal issuance (e.g., publication) of the resource.
Creator
An entity primarily responsible for making the resource
Administración General de Puertos
Type
The nature or genre of the resource
Disposición
Abstract
A summary of the resource.
Se establecen medidas conducentes para dictar un protocolo de actuación que, en el ámbito del TRANSPORTE FLUVIAL y MARÍTIMO LACUSTRE, permita controlar la propagación del CORONAVIRUS (COVID-19), en jurisdicción de esta ADMINISTARACIÓN GENERAL DE PUERTOS SOCIEDAD DEL ESTADO
-
https://biblioteca.senasa.gob.ar/files/original/162dc73c47908ee0d9d1dc6abf38833c.pdf
ef48f922f21018827af9f1c113edd635
PDF Text
Text
https://www.boletinoficial.gob.ar/#!DetalleNorma/226735/20200314
ADMINISTRACIÓN NACIONAL DE LA SEGURIDAD SOCIAL
Resolución 70/2020
RESOL-2020-70-ANSES-ANSES
Ciudad de Buenos Aires, 13/03/2020
VISTO el Expediente N° EX-2020-16870929-ANSES-DPAYT#ANSES del Registro de esta ADMINISTRACIÓN
NACIONAL DE LA SEGURIDAD SOCIAL (ANSES), el Decreto N° DECNU-2020-260-APN-PTE de fecha 12 de
marzo de 2020, y
CONSIDERANDO:
Que con fecha 11 de marzo de 2020, la ORGANIZACIÓN MUNDIAL DE LA SALUD (OMS) declaró el brote del
nuevo coronavirus como una pandemia, luego de que el número de personas infectadas por COVID-19 a nivel
global llegara a 118.554, y el número de muertes a 4.281, afectando hasta ese momento a 110 países.
Que mediante el Decreto de Necesidad y Urgencia citado en el VISTO, el Poder Ejecutivo Nacional amplió la
emergencia pública en materia sanitaria establecida por Ley N° 27.541, en virtud de la pandemia declarada por la
ORGANIZACIÓN MUNDIAL DE LA SALUD (OMS) en relación con el coronavirus COVID-19, por el plazo de UN (1)
año a partir de la entrada en vigencia del mencionado decreto.
Que en el marco de la emergencia sanitaria y de la situación epidemiológica actual, resulta necesario implementar
acciones y políticas excepcionales para el adecuado cumplimiento de las recomendaciones dispuestas por el
Gobierno Nacional.
Que en el sentido expuesto en el considerando precedente, deviene necesario que las áreas de atención al público
de esta Administración Nacional, cuenten con un esquema que regule la asistencia presencial a fin de evitar
aglomeración de personas, para mitigar la propagación del coronavirus COVID-19.
Que asimismo, se adoptarán medidas tendientes a fortalecer el resguardo de los grupos de riesgo, garantizando el
ejercicio de sus derechos en el marco sanitario existente.
Que en el presente contexto, y con iguales fines, se implementarán acciones tendientes a resguardar las
condiciones de seguridad e higiene de los trabajadores y trabajadoras de este Organismo.
Que el Servicio Jurídico Permanente ha tomado la intervención de su competencia.
Que la presente Resolución se dicta en uso de las facultades conferidas por el artículo 3° del Decreto N° 2741/91,
el artículo 36 de la Ley N° 24.241 y el Decreto N° 35/19.
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Por ello,
EL DIRECTOR EJECUTIVO DE LA ADMINISTRACION NACIONAL DE LA SEGURIDAD SOCIAL
RESUELVE:
ARTÍCULO 1°.- Establécese que, a partir del día 17 de marzo y hasta el día 15 de abril del año 2020, las áreas de
atención al público de ANSES contarán con un esquema reducido de atención al público, en virtud de la pandemia
declarada por la ORGANIZACIÓN MUNDIAL DE LA SALUD (OMS) en relación al coronavirus COVID-19, a fin de
mitigar su propagación y su impacto sanitario.
ARTÍCULO 2°.- Dispónese que durante el período citado en el ARTÍCULO precedente, sólo se atenderá en los
diferentes centros de atención, al público que cuente con turno previo asignado.
ARTÍCULO 3°.- Establécese que los días hábiles comprendidos en el período citado en el ARTÍCULO 1°, no serán
computados a los fines de los plazos procesales administrativos.
ARTÍCULO 4°.- Instrúyase a la Dirección General de Diseño de Normas y Procesos para que, conjuntamente con
las áreas competentes, establezca los procedimientos que resulten necesarios para implementar lo dispuesto en la
presente Resolución.
ARTÍCULO 5°.- Establécese que el plazo establecido en el ARTÍCULO 1° de la presente Resolución podrá tener
modificaciones según la evolución de la situación epidemiológica.
ARTÍCULO 6°.- Comuníquese, publíquese, dése a la Dirección Nacional del Registro Oficial y archívese. Alejandro
Vanoli Long Biocca
e. 14/03/2020 N° 14860/20 v. 14/03/2020
Fecha de publicación 14/03/2020
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�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Otros organismos
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Resolución ANSES N° 0070/2020
Description
An account of the resource
ANSES implementa un esquema reducido de atención al público
.
Source
A related resource from which the described resource is derived
<a href="http://servicios.infoleg.gob.ar/infolegInternet/verNorma.do?id=335472">Resolución ANSES N° 0070/2020</a>
Language
A language of the resource
Es
Type
The nature or genre of the resource
Resolución
Date
A point or period of time associated with an event in the lifecycle of the resource
Viernes 13 de Marzo de 2020
Creator
An entity primarily responsible for making the resource
Administración Nacional de Seguridad Social
Abstract
A summary of the resource.
Se establece que a partir del día 17 de marzo y hasta el día 15 de abril del año 2020, las áreas de atención al público de ANSES contarán con un esquema reducido de atención al público, en virtud de la pandemia declarada por la ORGANIZACIÓN MUNDIAL DE LA SALUD (OMS) en relación al coronavirus COVID-19, a fin de mitigar su propagación y su impacto sanitario. Dicho plazo podrá tener modificaciones según la evolución de la situación epidemiológica
-
https://biblioteca.senasa.gob.ar/files/original/9565d0ff3694f757f54418f300acbc1d.pdf
85e3b8931852ad72676f6de2d1ca28e1
PDF Text
Text
https://www.boletinoficial.gob.ar/#!DetalleNorma/227054/20200320
ADMINISTRACIÓN NACIONAL DE LA SEGURIDAD SOCIAL
Resolución 79/2020
RESOL-2020-79-ANSES-ANSES
Ciudad de Buenos Aires, 19/03/2020
VISTO el Expediente N° EX-2020-18091302-ANSES-DPB#ANSES, las Resoluciones D.E.-N. N° 567 de fecha 30
de diciembre de 2013, y N° 648 de fecha 11 de diciembre de 2014, y
CONSIDERANDO:
Que con fecha 11 de marzo de 2020, la ORGANIZACIÓN MUNDIAL DE LA SALUD (OMS) declaró el brote del
nuevo coronavirus COVID-19 como una pandemia.
Que a través del artículo 1° del Decreto de Necesidad y Urgencia Nº 260/2020 se amplió la emergencia pública en
materia sanitaria establecida por Ley N° 27.541.
Que en dicho marco esta ADMINISTRACION NACIONAL DE LA SEGURIDAD SOCIAL, con el objetivo de
coadyuvar a que el grupo vulnerable de adultos mayores jubilados y pensionados permanezca en sus hogares, ha
instado a todos los agentes pagadores de jubilaciones y pensiones a tener por cumplido el trámite de “fe de vida”
con las modalidades hasta ahora vigentes y además con la posibilidad de presentación de una declaración jurada
suscripta por el beneficiario y cuyo original sea rubricado por persona humana con capacidad legal para
responsabilizarse, quien deberá acreditar su identidad y suscribir la documentación pertinente por ante el agente de
pago, de acuerdo a las exigencias de cada entidad.
Que la acelerada propagación del virus torna ineludible tomar medidas tendientes a proteger a la población de un
posible contagio y circulación del virus.
Que resulta necesario tomar medidas excepcionales y urgentes a fin de minimizar los riesgos de la salud pública,
en concordancia con las medidas dispuestas por el Estado Nacional.
Que en dicho marco esta ADMINISTRACION NACIONAL DE LA SEGURIDAD SOCIAL ha merituado pertinente
reforzar las medidas con el objetivo de evitar el contacto personal de la población y el resguardo de los grupos de
riesgo suspendiendo el trámite de fe de vida, a efectos de garantizar el inmediato cobro de las prestaciones
previsionales puestas al pago durante los meses de marzo y abril del año en curso.
Que las Resoluciones D.E.-N. N° 567 de fecha 30 de diciembre de 2013, y N° 648 de fecha 11 de diciembre de
2014 detallan el procedimiento de pago de las prestaciones a cargo de la ADMINISTRACIÓN NACIONAL DE LA
SEGURIDAD SOCIAL (ANSES) y de aquellas que pone al pago por cuenta y orden de terceros.
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Que la Dirección General de Finanzas y la Subdirección Ejecutiva de Administración de esta Administración
Nacional han tomado la intervención de acuerdo a sus competencias.
Que el Servicio Jurídico Permanente de esta Administración Nacional ha tomado debida intervención de acuerdo a
sus competencias.
Que la presente se dicta en uso de las facultades conferidas por el artículo 36 de la Ley Nº 24.241, el artículo 3° del
Decreto Nº 2.741/91 y el Decreto Nº 35/19.
Por ello,
EL DIRECTOR EJECUTIVO DE LA ADMINISTRACIÓN NACIONAL DE LA SEGURIDAD SOCIAL
RESUELVE:
ARTICULO 1°.- Suspéndase el trámite de actualización de fe de vida por parte de los jubilados y pensionados del
Sistema Integrado Previsional Argentino (SIPA) y Pensiones No Contributivas a efectos de garantizarles el cobro de
las prestaciones puestas al pago durante los meses de marzo y abril de 2020, por los motivos expuestos en los
considerandos de la presente.
ARTICULO 2°.- Déjase establecido que las Entidades Pagadoras conservan la responsabilidad de rendir como
impagos, en el marco de las operatorias vigentes, los fondos correspondientes, luego del fallecimiento del titular del
beneficio y a partir de la recepción de la novedad de fallecidos informada por esta ANSES, para los mensuales de
marzo y abril de 2020.
ARTICULO 3°.- Establécese que la presente medida entrará en vigencia en el día de su publicación en el BOLETIN
OFICIAL.
ARTICULO 4°.- Comuníquese, publíquese, dese a la DIRECCIÓN NACIONAL DEL REGISTRO OFICIAL y
oportunamente, archívese. Alejandro Vanoli Long Biocca
e. 20/03/2020 N° 15885/20 v. 20/03/2020
Fecha de publicación 20/03/2020
2 de 2
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Otros organismos
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Resolución ANSES N° 0079/2020
Description
An account of the resource
Jubilados y pensionados: se suspende el trámite de supervivencia
Source
A related resource from which the described resource is derived
<a href="http://servicios.infoleg.gob.ar/infolegInternet/verNorma.do?id=335753">Resolución ANSES N° 0079/2020</a>
Type
The nature or genre of the resource
Resolución
Date
A point or period of time associated with an event in the lifecycle of the resource
Jueves 19 de Marzo de 2020
Date Available
Date (often a range) that the resource became or will become available.
Creator
An entity primarily responsible for making the resource
Administración Nacional de Seguridad Social
Language
A language of the resource
Es
Abstract
A summary of the resource.
Se suspende el trámite de actualización de fe de vida por parte de los jubilados y pensionados del Sistema Integrado Previsional Argentino (SIPA) y Pensiones No Contributivas a efectos de garantizarles el cobro de las prestaciones puestas al pago durante los meses de marzo y abril de 2020.
-
https://biblioteca.senasa.gob.ar/files/original/e0ec44bad79375ad7d4d10a8e9032724.pdf
4da859907abb45d724f0fcf8cb6ce445
PDF Text
Text
https://www.boletinoficial.gob.ar/#!DetalleNorma/226928/20200318
AGENCIA NACIONAL DE DISCAPACIDAD
Resolución 60/2020
RESOL-2020-60-APN-DE#AND
Ciudad de Buenos Aires, 17/03/2020
VISTO el Expediente N° EX-2020-16517661-APN-DE#AND, las Leyes Nros. 13.478, 19.279, 22.431, 24.901 y
27.541 y sus modificatorias y complementarias, los Decretos Nros. 1313/93, 1193/98, 806/1, 698/17, 95/18, 160/18
y 260/20, la Resolución N° 675/09 del MINISTERIO DE SALUD, la Resolución N° 5/15 del ORGANO DE CONTROL
DE CONCESIONES VIALES y las Resoluciones Nros. 39/18 y 8/20 de la AGENCIA NACIONAL DE
DISCAPACIDAD, y
CONSIDERANDO:
Que por el Decreto N° 698 de fecha 5 de septiembre de 2017 se creó la AGENCIA NACIONAL DE
DISCAPACIDAD, como organismo descentralizado en la órbita de la SECRETARIA GENERAL de la
PRESIDENCIA DE LA NACION, encargado del diseño, coordinación y ejecución general de las políticas públicas
en materia de discapacidad, la elaboración y ejecución de acciones tendientes a promover el pleno ejercicio de los
derechos de las personas en situación de discapacidad y la conducción del proceso de otorgamiento de las
pensiones por invalidez y las emergentes de las Leyes N° 25.869 y N° 26.928.
Que por el Decreto N° 95 del 1° de febrero de 2018 se suprimió el SERVICIO NACIONAL DE REHABILITACION y
se transfirió a la órbita de la AGENCIA NACIONAL DE DISCAPACIDAD, la que será continuadora a todos los
efectos legales del precitado SERVICIO NACIONAL DE REHABILITACION.
Que por el Decreto N° 160 de fecha 27 de febrero de 2018 se transfirió al ámbito de la AGENCIA NACIONAL DE
DISCAPACIDAD el PROGRAMA FEDERAL DE SALUD INCLUIR SALUD.
Que por la Ley Nº 24.901, sus modificatorias y complementarias, se instituye un sistema de prestaciones básicas
de atención integral a favor de las personas con discapacidad, contemplando acciones de prevención, asistencia,
promoción y protección, con el objeto de brindarles una cobertura integral a sus necesidades y requerimientos.
Que por el artículo 3º de la Ley Nº 22.431, modificado por el artículo 8° del Decreto Nº 95/2018, se establece que la
AGENCIA NACIONAL DE DISCAPACIDAD certificará en cada caso la existencia de la discapacidad, su naturaleza
y su grado, así como las posibilidades de rehabilitación del afectado e indicará, teniendo en cuenta la personalidad
y los antecedentes del afectado, que tipo de actividad laboral o profesional puede desempeñar, añandiendo que el
certificado que se expida se denominará Certificado Único de Discapacidad (CUD).
Que el artículo 10° de la Ley N° 24.901 determina que a los efectos de dicha ley, la discapacidad deberá
acreditarse conforme a lo establecido por el artículo 3° de la Ley N° 22.431 y por leyes provinciales análogas.
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Que el artículo 10° del Anexo I del Decreto N° 1193 de fecha 8 de octubre de 1998 -reglamentario de la Ley N°
24.901- determina que el certificado de discapacidad se otorgará previa evaluación del beneficiario por un equipo
interdisciplinario que se constituirá a tal fin y comprenderá el diagnóstico funcional y la orientación prestacional,
información que se incorporará al Registro Nacional de Personas con Discapacidad.
Que mediante la Resolución Nº 675 de fecha 12 de mayo de 2009 del MINISTERIO DE SALUD DE LA NACION,
modificatorias y complementarias, se aprueba el Modelo del CUD creado por el artículo 3º de la Ley Nº 22.431, que
prevé una vigencia y fecha de vencimiento, conforme la evaluación de la Junta Evaluadora Interdisciplinaria.
Que por el artículo 12° de la Ley Nº 19.279, reglamentado por el artículo 17 del Decreto N° 1313 de fecha 24 de
junio de 1993, se adopta el Símbolo Internacional de Acceso que, en otros fines, se utiliza para acreditar el derecho
a la franquicia de libre tránsito y estacionamiento.
Que por la Resolución Nº 5 de fecha 28 de enero de 2015 del ORGANO DE CONTROL DE CONCESIONES
VIALES se aprueba el Reglamento de exención de peaje para personas con discapacidad y se establece que los
usuarios que deseen acogerse al beneficio deben presentar, entre otra documentación, el Certificado Único de
Discapacidad vigente y el Símbolo Internacional de Acceso.
Que el Símbolo Internacional de Acceso, la Exención de pago de peaje, así como el troquel para pase de transporte
púbico destinados a personas con discapacidad poseen una fecha de vencimiento sujeta a la de de expiración del
Certificado Unico de Discapacidad.
Que el Certificado Unico de Discapacidad es un documento que certifica la discapacidad de la persona y le permite
acceder a derechos y prestaciones que brinda el Estado Nacional en materia de salud, transporte, asignaciones
familiares, excensión de impuestos, entre otros.
Que por la Resolución N° 39 del 31 de enero de 2019 de la AGENCIA NACIONAL DE DISCAPACIDAD se aprobó
un nuevo Formulario Certificado Médico Oficial (CMO) que deben presentar los solicitantes de Pensiones no
Contributivas por Invalidez instituidas en el artículo 9° de la Ley N° 13.478.
Que por la Resolución Nº 8 del 28 de enero de 2020 de la AGENCIA NACIONAL DE DISCAPACIDAD se establece
que, hasta tanto el Certificado Médico Oficial (CMO) Digital no resulte accesible digitalmente en todas las provincias
de la República Argentina, se garantizará el inicio del trámite correspondiente a la solicitud de una Pensión no
Contributiva por Invalidez, a través de las Unidades de Atención Integral (UDAI) de la ADMINISTRACION
NACIONAL DE LA SEGURIDAD SOCIAL (ANSeS), conforme el Convenio de colaboración identificado como
CONVE-2018-43706986-ANSES-ANSES, aún cuando, en esa instancia inicial, no se acompañe el respectivo CMO.
Que es de público y notorio conocimiento la situación excepcional derivada de la crítica situación sanitaria
provocada por el virus COVID-19 cuya propagación a nivel mundial pone en riesgo a la población.
Que el Gobierno Nacional debe garantizar los derechos esenciales de la población y su goce efectivo, siendo un
interés prioritario tener asegurado el acceso sin restricciones a la salud, seguridad e intereses económicos,
conforme al artículo 42 de la Constitución Nacional.
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Que mediante Decreto Nº 260 de fecha 12 de marzo de 2020 se amplía la emergencia pública en materia sanitaria
establecida por Ley N° 27.541, en virtud de la Pandemia declarada por la ORGANIZACIÓN MUNDIAL DE LA
SALUD (OMS) en relación con el coronavirus COVID-19, por el plazo de UN (1) año a partir de su entrada en
vigencia.
Que dentro de las competencias que la AGENCIA NACIONAL DE DISCAPACIDAD, resulta necesario implementar
medidas direccionadas a coadyuvar con el esfuerzo sanitario para evitar la propagación de la enfermedad.
Que a los fines de asegurar la protección sanitaria, evitar situaciones de contagio y aglomeraciones se estima
necesario suspender, hasta el 31 de marzo de 2020, algunas prestaciones básicas de atención integral a favor de
las personas con discapacidad de la Ley Nº 24.901, en el marco del PROGRAMA FEDERAL DE SALUD INCLUIR
SALUD, con excepción de los sistemas alternativos al grupo familiar y de los Centros de Rehabilitación, así como
garantizar la continuidad de todas las prestaciones alimentarias que se brinden.
Que, asimismo, deviene necesario prorrogar los plazos de vigencia de los Certificados Únicos de Discapacidad
(CUD), del correspondiente troquel de pase de transporte público y del Símbolo Internacional de Acceso y ratificar
la posibilidad de iniciar trámites de solicitud de Pensiones no Contributivas por Invalidez aún cuando no fuere
posible la obtención del Certificado Médico Oficial (CMO), ya sea digital o en formato papel.
Que la DIRECCION GENERAL DE ASUNTOS JURIDICOS de la SECRETARIA GENERAL de la PRESIDENCIA
DE LA NACIONA ha tomado la intervención de su competencia.
Que la presente se dicta en uso de las facultades conferidas por los Decretos N° 698/2017, 868/17, 160/18 y N°
70/20.
Por ello,
EL DIRECTOR EJECUTIVO DE LA AGENCIA NACIONAL DE DISCAPACIDAD
RESUELVE:
ARTICULO 1º.- Suspéndanse, hasta el 31 de marzo de 2020, las prestaciones básicas de atención integral a favor
de las personas con discapacidad de la Ley Nº 24.901, sus modificatorias y complementarias, del PROGRAMA
FEDERAL DE SALUD INCLUIR SALUD que a continuación se mencionan: Centros de día; Centros educativos
terapéuticos, Centros de formación laboral, Aprestamiento laboral, Escolaridad Inicial, Educación general básica,
Centros de rehabilitación ambulatorios, Prestaciones de consultorio, Servicios de estimulación temprana en
consultorio y a domicilio, Prestaciones de apoyo escolar, Módulo de maestro de apoyo, Módulo de apoyo a la
integración escolar, Escuelas especiales y Transporte; en todas sus modalidades.
Durante el período que dure la suspensión establecida por el ARTICULO 1º de la presente, los centros
correspondientes deberán garantizar la continuidad de todas las prestaciones alimentarias que se brinden, en lo
posible mediante entrega de viandas, y en caso que se mantuvieran en funcionamiento los comedores, deberán
observarse las disposiciones de higiene y salubridad, sobre distancias mínimas y toda otra que la autoridad
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sanitaria disponga durante este período excepcional.
ARTICULO 2º.- Se sugiere, en orden a la responsabilidad social, que los transportistas que conforme el ARTICULO
1º de la presente no brindarán prestación alguna durante el período de suspensión, se pongan a disposicion de los
centros que presten servicios de alimentación para colaborar en la entrega de las viandas alimentarias.
ARTICULO 3º.- Establécese que, sin perjuicio de las prestaciones suspendidas por el ARTICULO 1º de la presente,
los sistemas alternativos al grupo familiar previstos en la Ley Nº 24.901, sus modificatorias y complementarias,
entendiéndose por tales a: residencias, pequeños hogares y hogares, con prestaciones combinadas, continuarán
prestando exclusivamente los servicios de vivienda, alimentación y atención personalizada.
Del mismo modo, continuarán prestando servicios los Centros de rehabilitación con internación, manteniendo todas
las prestaciones habituales, con excepción de las suspendidas por el ARTICULO 1º de la presente.
ARTICULO 4º.- Todas las prestaciones que se suspenden por el ARTICULO 1º de la presente serán abonadas por
el PROGRAMA FEDERAL DE SALUD DE INCLUIR SALUD, en la forma y de acuerdo con los procedimientos
administrativos correspondientes.
ARTICULO 5º.- Prorrógase la vigencia de los plazos de vencimiento del Certificado Unico de Discapacidad (CUD),
del correspondiente troquel de pase de transporte público y del Símbolo Internacional de Acceso, por un plazo de
NOVENTA (90) días corridos a partir de la entrada en vigencia de la presente, y de aquellos cuyo vencimiento
operó a partir del 16 de febrero de 2020.
Por la DIRECCION NACIONAL DE POLITICAS Y REGULACION DE SERVICIOS, póngase en conocimiento del
contenido del presente artículo a la SUPERINTENDENCIA DE SERVICIOS DE SALUD DE LA NACION, a la
COMISION NACIONAL DE REGULACION DEL TRANSPORTE y al ORGANO DE CONTROL DE CONCESIONES
VIALES.
ARTICULO 6º.- Establécese que las juntas evaluadoras continuarán su funcionamiento, con guardias de
emergencia, para la obtención del Certificado Unico de Discapacidad (CUD) por primera vez, según lo requierean
las personas con discapacidad.
Instrúyase a la DIRECCION NACIONAL DE POLITICAS Y REGULACION DE SERVICIOS a los fines de poner en
conocimiento lo dispuesto por el presente artículo a todas las Provincias y a la Ciudad Autónoma de Buenos Aires.
ARTICULOS 7º.- Establécese la posibilidad de iniciar todo trámite en el que se requiera el Certificado Médico Oficial
(CMO) o CMO Digital, a través del Trámite a Distancia (TAD) de la ADMINISTRACION NACIONAL DE LA
SEGURIDAD SOCIAL (ANSeS) o los Centros de Atención Local de ANDIS, el cual podrá presentarse con
posterioridad a los NOVENTA (90) días corridos de la presentación.
Instrúyase a la DIRECCION NACIONAL DE APOYOS Y ASIGNACIONES ECONOMICAS a los fines de la
implementación de lo dispuesto en el presente artículo y para que ponga en conocimiento a la ADMINISTRACION
NACIONAL DE LA SEGURIDAD SOCIAL de los términos de la presente.
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ARTICULO 8º.- Conforme lo establecido por la Ley Nº 24.901, sus normas modificatorias y complementarias,
convócase en forma urgente para el día jueves 19 de marzo de 2020 a las 11 hs. en la sede de Dragones 2201,
Pabellón principal, Salón Blanco, de la Cudad Autónoma de Buenos Aires, una reunión urgente del Directorio del
Sistema de Prestaciones Básicas de Atención Integral de Personas con Discapacidad, con el fin de tratar los
efectos de la emergencia santaria sobre el sistema de prestaciones básicas para personas con discapacidad.
Para ello, instrúyase a la Secretaría General de la AGENCIA NACIONAL DE DISCAPACIDAD para que en forma
inmediata y urgente proceda a la convocatoria de los miembros del Directorio.
ARTICULO 9º.- La presente medida entrará en vigencia a partir de su publicación.
ARTICULO 10º.- Comuníquese, publíquese, dése a la DIRECCION NACIONAL DEL REGISTRO OFICIAL, y
oportunamente, archívese. Claudio Flavio Augusto Esposito
e. 18/03/2020 N° 15583/20 v. 18/03/2020
Fecha de publicación 18/03/2020
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Title
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Otros organismos
Dublin Core
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Title
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Resolución AND N° 0060/2020
Description
An account of the resource
Suspensión transitoria de algunas prestaciones básicas a personas con discapacidad
Source
A related resource from which the described resource is derived
<a href="http://servicios.infoleg.gob.ar/infolegInternet/verNorma.do?id=335626">Resolución AND N° 0060/2020</a>
Language
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Es
Type
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Resolución
Date
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Martes 17 de Marzo de 2020
Date Issued
Date of formal issuance (e.g., publication) of the resource.
Publicada en el Boletín Oficial del 18-mar-2020, Número: 34332, Página: 20
Abstract
A summary of the resource.
Se suspende hasta el 31 de marzo de 2020, las prestaciones básicas de atención integral a favor de las personas con discapacidad de la Ley Nº 24.901, sus modificatorias y complementarias, del PROGRAMA FEDERAL DE SALUD INCLUIR SALUD que a continuación se mencionan: Centros de día; Centros educativos terapéuticos, Centros de formación laboral, Aprestamiento laboral, Escolaridad Inicial, Educación general básica, Centros de rehabilitación ambulatorios, Prestaciones de consultorio, Servicios de estimulación temprana en consultorio y a domicilio, Prestaciones de apoyo escolar, Módulo de maestro de apoyo, Módulo de apoyo a la integración escolar, Escuelas especiales y Transporte; en todas sus modalidades.
Se prorroga la vigencia de los plazos de vencimiento del Certificado Unico de Discapacidad (CUD), del correspondiente troquel de pase de transporte público y del Símbolo Internacional de Acceso, por un plazo de NOVENTA (90) días corridos.
Se convoca para el día jueves 19 de marzo de 2020, una reunión urgente del Directorio del Sistema de Prestaciones Básicas de Atención Integral de Personas con Discapacidad, con el fin de tratar los efectos de la emergencia sanitaria sobre el sistema de prestaciones básicas para personas con discapacidad.
Creator
An entity primarily responsible for making the resource
Agencia Nacional de Discapacidad
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https://biblioteca.senasa.gob.ar/files/original/91150cd28b1402a394580addf8298bca.pdf
3b40410d8381cb6c66eed3cc84e899f2
PDF Text
Text
https://www.boletinoficial.gob.ar/#!DetalleNorma/227055/20200320
AGENCIA NACIONAL DE DISCAPACIDAD
Resolución 63/2020
RESOL-2020-63-APN-DE#AND
Ciudad de Buenos Aires, 19/03/2020
VISTO el Expediente N° EX-2020-16518599-APN-DE#AND, las Leyes Nros. 22.431, 24.901, sus modificatorias y
complementarias, los Decretos Nros. 762/97, 1193/98, 698/17, 95/18, 160/18 y 260/20, y CONSIDERANDO: Que,
por el artículo 3° de la Ley N° 22.431, sustituido por el Decreto de Necesidad y Urgencia N° 95 de fecha 1° de
febrero de 2018, se establece que la AGENCIA NACIONAL DE DISCAPACIDAD certificará la existencia de la
discapacidad mediante la expedición de un Certificado Único de Discapacidad (CDU), plenamente valido en todo el
territorio nacional, en todos los supuestos en que sea necesario invocarla, salvo lo dispuesto en el artículo 19 de la
mencionada Ley.
Que, asimismo, el mencionado artículo establece que idéntica validez, en cuanto a sus efectos, tendrán los
certificados emitidos por las provincias adheridas a la Ley Nº 24.901, previo cumplimiento de los requisitos y
condiciones que se establezcan por reglamentación.
Que, por el Decreto reglamentario Nº 1193 de fecha 8 de octubre de 1998 se establece que la COMISION
NACIONAL ASESORA PARA LA INTEGRACION DE LAS PERSONAS DISCAPACITADAS será el organismo
regulador del “Sistema de Prestaciones Básicas de Atención Integral a favor de las Personas con Discapacidad” y,
contará para su administración con un DIRECTORIO DEL SISTEMA DE PRESTACIONES BASICAS DE
ATENCION INTEGRAL A FAVOR DE LAS PERSONAS CON DISCAPACIDAD, cuya presidencia será ejercida por
el Presidente de la referida Comisión Nacional.
Que por el Decreto N° 698 de fecha 5 de septiembre de 2017 se creó la AGENCIA NACIONAL DE
DISCAPACIDAD, como organismo descentralizado en la órbita de la SECRETARIA GENERAL de la
PRESIDENCIA DE LA NACION, encargado del diseño, coordinación y ejecución general de las políticas públicas
en materia de discapacidad, la elaboración y ejecución de acciones tendientes a promover el pleno ejercicio de los
derechos de las personas en situación de discapacidad y la conducción del proceso de otorgamiento de las
pensiones por invalidez y las emergentes de las Leyes N° 25.869 y N° 26.928.
Que, por otra parte, la norma antes referida, estableció que la AGENCIA NACIONAL DE DISCAPACIDAD será el
organismo continuador, a todos los fines, de las ex COMISIÓN NACIONAL DE PENSIONES ASISTENCIALES y ex
COMISIÓN NACIONAL ASESORA PARA LA INTEGRACIÓN DE LAS PERSONAS CON DISCAPACIDAD.
Que por el Decreto N° 95 del 1° de febrero de 2018 se suprimió el SERVICIO NACIONAL DE REHABILITACION y
se transfirió a la órbita de la AGENCIA NACIONAL DE DISCAPACIDAD, la que será continuadora a todos los
efectos legales del precitado SERVICIO NACIONAL DE REHABILITACION.
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Que por el Decreto N° 160 de fecha 27 de febrero de 2018 se transfirió al ámbito de la AGENCIA NACIONAL DE
DISCAPACIDAD el PROGRAMA FEDERAL DE SALUD INCLUIR SALUD. Que es de público y notorio
conocimiento la situación excepcional derivada de la crítica situación sanitaria provocada por el virus COVID-19
cuya propagación a nivel mundial pone en riesgo a la población.
Que mediante Decreto Nº 260 de fecha 12 de marzo de 2020, normas modificatorias y complementarias, se amplía
la emergencia pública en materia sanitaria establecida por Ley N° 27.541, en virtud de la Pandemia declarada por la
ORGANIZACIÓN MUNDIAL DE LA SALUD (OMS) en relación con el COVID-19, por el plazo de UN (1) año a partir
de su entrada en vigencia.
Que mediante reunión extraordinaria, convocada por el artículo 8° de la Resolución N° 60 de fecha 17 de marzo de
2020 de la AGENCIA NACIONAL DE DISCAPACIDAD, del DIRECTORIO DEL SISTEMA DE PRESTACIONES
BASICAS DE ATENCION INTEGRAL A FAVOR DE LAS PERSONAS CON DISCAPACIDAD del día 19 de marzo
de 2020, de la que da cuenta mediante Acta N° 392, identificada como IF2020-18075533-APN-DE#AND, éste
resolvió adoptar medidas a los fines de asegurar la protección sanitaria a favor de las personas con discapacidad.
Que la presente medida se dicta en ejercicio de las funciones reconocidas al DIRECTORIO DEL SISTEMA DE
PRESTACIONES BASICAS DE ATENCION INTEGRAL A FAVOR DE LAS PERSONAS CON DISCAPACIDAD por
el artículo 5º apartados a) y e) del Anexo A del Decreto 1193/98.
Por ello,
EL PRESIDENTE DEL DIRECTORIO DEL SISTEMA DE PRESTACIONES BASICAS DE ATENCION INTEGRAL
A FAVOR DE LAS PERSONAS CON DISCAPACIDAD
RESUELVE:
ARTICULO 1º.- Suspéndanse, por criterios epidemiológicos, hasta el 31 de marzo de 2020, las prestaciones
básicas de atención integral a favor de las personas con discapacidad de la Ley Nº 24.901, sus modificatorias y
complementarias, que a continuación se mencionan: Centros Educativos Terapéuticos, Centros de Día, Servicios
de Rehabilitación, Servicios de Apoyo a la Inclusión Educativa y modalidades de prestaciones de apoyo.
ARTICULO 2°. Se recomienda que los Hogares y Residencias previstos en la Ley N° 24.901, sus modificatorias y
complementarias, extremen las medidas higiénicas para evitar la propagación del COVID-19 y reducir la circulación
de Profesionales de actividades no esenciales.
ARTICULO 3°.- Establécese que las instituciones mencionadas en el ARTICULO 2° de la presente continuaran
prestando exclusivamente los servicios de vivienda, alimentación y atención personalizada.
ARTICULO 4°.- Prorrógase, por un plazo de NOVENTA (90) días corridos, la vigencia de los plazos de vencimiento
de los Certificados Únicos de Discapacidad (CUD), y de los aquellos emitidos por las provincias adheridas a la Ley
Nº 24.901 (NO CUD), cuyo vencimiento opere en los NOVENTA (90) días posteriores a la publicación de la
presente. Asimismo, prorrógase, por un plazo de CIENTO (120) días corridos, la vigencia de los Certificados Únicos
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de Discapacidad (CUD), y de los aquellos emitidos por las provincias adheridas a la Ley Nº 24.901 (NO CUD), cuyo
vencimiento hubiera operado en los TREINTA (30) días corridos anteriores a la publicación de la presente.
ARTICULO 5°.- En razón de la adopción de las medidas mencionadas, se insta a la SUPERINTENDENCIA DE
SERVICIOS DE SALUD y al INSTITUTO NACIONAL DE SERVICIOS PARA JUBILADOS Y PENSIONADOS
(INSJP), para que emitan los actos administrativos y comunicaciones pertinentes, a fin garantizar la cobertura de
las prestaciones médico asistenciales que se vean afectadas por esta medida y se encuentren previstas en el
Nomenclador de Prestaciones Básicas con Discapacidad, conforme Resolución N° 428/99 del ex MINISTERIO DE
SALUD Y ACCION SOCIAL.
ARTICULO 6º.- La presente medida entrará en vigencia a partir de su publicación y será plausible de
modificaciones de acuerdo a las decisiones que adopte el Gobierno Nacional en relación al COVID-19.
ARTICULO 7°.- Invítase a las autoridades de las provincias y de la Ciudad Autónoma de Buenos Aires a la
adopción de medidas similares a la presente.
ARTICULO 8º.- Comuníquese, publíquese, dése a la DIRECCION NACIONAL DEL REGISTRO OFICIAL, y
oportunamente, archívese Claudio Flavio Augusto Esposito
e. 20/03/2020 N° 15884/20 v. 20/03/2020
Fecha de publicación 20/03/2020
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�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Otros organismos
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Resolución AND N° 0063/2020
Description
An account of the resource
Suspensión transitoria de algunas prestaciones básicas a personas con discapacidad
Source
A related resource from which the described resource is derived
<a href="http://servicios.infoleg.gob.ar/infolegInternet/verNorma.do?id=335754">Resolucion AND N° 0063/2020</a>
Date
A point or period of time associated with an event in the lifecycle of the resource
Jueves 19 de Marzo de 2020
Language
A language of the resource
Es
Creator
An entity primarily responsible for making the resource
Agencia Nacional de Discapacidad
Type
The nature or genre of the resource
Resolución
Abstract
A summary of the resource.
Se suspende, por criterios epidemiológicos, hasta el 31 de marzo de 2020, las prestaciones básicas de atención integral a favor de las personas con discapacidad de la Ley Nº 24.901, sus modificatorias y complementarias, que a continuación se mencionan: Centros Educativos Terapéuticos, Centros de Día, Servicios de Rehabilitación, Servicios de Apoyo a la Inclusión Educativa y modalidades de prestaciones de apoyo.
Se prorroga, por un plazo de NOVENTA (90) días corridos, la vigencia de los plazos de vencimiento de los Certificados Únicos de Discapacidad (CUD), y de los aquellos emitidos por las provincias adheridas a la Ley Nº 24.901 (NO CUD), cuyo vencimiento opere en los NOVENTA (90) días posteriores a la publicación de la presente. Asimismo, prorrógase, por un plazo de CIENTO (120) días corridos, la vigencia de los Certificados Únicos de Discapacidad (CUD), y de los aquellos emitidos por las provincias adheridas a la Ley Nº 24.901 (NO CUD), cuyo vencimiento hubiera operado en los TREINTA (30) días corridos anteriores a la publicación de la presente.
-
https://biblioteca.senasa.gob.ar/files/original/793a49ff283fe0faf2de722c186ed09c.pdf
deaf1f4ebb8c3d25ab4ceffdf3449c01
PDF Text
Text
Proceeding Paper
Isolation and Identification of Culturable Gut Microbiota in the
Larval Stage of Lesser Mealworm (Alphitobius diaperinus) †
Gisele Ivonne Antonuccio 1,2, *
1
2
3
*
†
Citation: Antonuccio, G.I.; Sauka,
D.H. Isolation and Identification of
Culturable Gut Microbiota in the
Larval Stage of Lesser Mealworm
and Diego Herman Sauka 1,3
Instituto de Microbiología y Zoología Agrícola (IMYZA), Instituto Nacional de Tecnología Agropecuaria (INTA),
Buenos Aires 1686, Argentina; sauka.diego@inta.gob.ar
Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA),
Ciudad Autónoma de Buenos Aires C1107ADR, Argentina
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),
Ciudad Autónoma de Buenos Aires C1425FQB, Argentina
Correspondence: antonuccio.gisele@inta.gob.ar
Presented at the 2nd International Electronic Conference on Microbiology, 1–15 December 2023; Available
online: https://ecm2023.sciforum.net.
Abstract: The highly prevalent pest Alphitobius diaperinus (Coleoptera: Tenebrionidae) causes significant structural damage in poultry farms. Despite previous investigations on its carriage of pathogenic
microorganisms, our understanding of its microbiome remains limited. This study aimed to analyze
the diversity of culturable gut microbiota in A. diaperinus obtained from laboratory breeding. Fifteen
seventh instar larvae underwent a 24-h starvation period, followed by surface disinfection. Dissected
midguts were homogenized and plated on nutrient agar (NA), brain heart infusion agar (BHI), and
Bacillus cereus agar (BC). The cultured isolates were subjected to gram staining, phylogenetic analysis,
biochemical property evaluation, and metabolic activity assessment. Bacterial counts were higher in
BHI (2.51 × 105 CFU/gut) than in NA (2.25 × 105 CFU/gut), possibly due to nutrient richness. NA
exhibited a dominant colony morphology of gram-negative bacilli, while BHI displayed additional
distinct colonies of gram-positive cocci. Surprisingly, yeast-like colonies were observed on BC plates.
Based on 16S rRNA gene sequences, eight bacterial isolates were identified as Enterobacter sp., and
two as Staphylococcus sp. Using RNA gene ITS region sequences, two yeast isolates were identified as
Debaryomyces sp. and Hyphopichia sp. A preliminary species-level identification of bacteria (Enterobacter cloacae, Staphylococcus gallinarum, and Staphylococcus succinus) was achieved using API systems
and complementary biochemical tests. Discrepancies between phylogenetic analysis and phenotypic
data suggest the potential existence of new species or subspecies. Further comprehensive studies are
required to confirm this hypothesis.
Keywords: Alphitobius diaperinus; gut microbiota; culturable microorganisms; bacterial diversity; yeast
(Alphitobius diaperinus) † . Biol. Life Sci.
Forum 2024, 31, 12. https://doi.org/
10.3390/ECM2023-16465
Academic Editor: Nico Jehmlich
Published: 30 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
1. Introduction
Alphitobius diaperinus, commonly known as the lesser mealworm, is an insect species
classified under the order Coleoptera and the family Tenebrionidae. This species, originally described by Panzer in 1797, has its origins in the African continent but has since
achieved a widespread global distribution. In addition to its significance in research as a
potential protein source for humans [1,2] and its role in exploring environmentally friendly
waste disposal solutions [3,4], A. diaperinus stands out as a notorious pest within poultry
environments.
Within the context of poultry farming, A. diaperinus presents a significant challenge
due to the substantial damage inflicted by both its larvae and adult individuals [5]. These
pests can cause extensive harm to poultry facilities, affecting not only the well-being of
the birds but also the economic viability of poultry production operations. Consequently,
4.0/).
Biol. Life Sci. Forum 2024, 31, 12. https://doi.org/10.3390/ECM2023-16465
https://www.mdpi.com/journal/blsf
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effective pest control strategies are essential to mitigate the negative impact of A. diaperinus
on the poultry industry.
Although previous investigations have explored the potential carriage of pathogenic
microorganisms by A. diaperinus, our understanding of its microbiome remains limited.
This knowledge gap is substantial because the insect’s gut microbiota can play a crucial
role in various aspects of its biology and ecology. Therefore, this study aims to examine
the diversity of culturable gut microbiota present in A. diaperinus specimens obtained from
laboratory rearing.
By shedding light on the microbiome of A. diaperinus, we aspire to contribute to a
better understanding of the biology of this pest and identify potential vulnerabilities that
can be targeted for more effective pest management in poultry facilities. Additionally, this
research may have broader implications for pest control strategies and could potentially
lead to more sustainable and environmentally friendly solutions for managing A. diaperinus
infestations in various agricultural settings.
2. Materials and Methods
2.1. Isolation and Count of Culturable Bacteria and Yeast from the Gut of A. diaperinus
The Instituto de Microbiología y Zoología Agrícola (IMYZA) at the Instituto Nacional
de Tecnología Agropecuaria (INTA) in Hurlingham, Buenos Aires, Argentina, has established a unique laboratory rearing system for A. diaperinus for research purposes, which, to
the best of our knowledge, is the only one of its kind in the country.
Fifteen seventh-instar larvae were subjected to a 24-h starvation period and then
underwent surface disinfection. This disinfection procedure involved a series of steps,
including immersion in 70% ethanol, exposure to a 15% bleach solution, and three rinses
in physiological solution, following the protocol outlined by Fang Lu et al. [6]. The third
rinse served as a control before proceeding with the dissection of the midguts. These
15 midguts were kept hydrated in physiological solution and subsequently homogenized in
a sterile mortar. The resulting midgut suspension underwent serial dilution. We inoculated
100 µL of four dilutions (ranging from 10−3 to 10−6 ), each in quintuplicate, on nutrient agar
(NA) and brain heart infusion agar (BHI). Additionally, 100 µL of the undiluted midgut
suspension was inoculated in triplicate on Bacillus cereus agar (BC). The plates were then
incubated for 72 h at 28 ◦ C. Selections of colonies displaying distinct morphologies were
isolated from the various culture media until pure cultures were obtained, facilitating
subsequent identification processes. The cultured isolates underwent plate counting of the
colonies to estimate the colony forming units (CFU) per gut, followed by Gram staining.
2.2. Bacterial and Yeast Identification
The isolated bacteria were identified at the genus level by amplifying and subsequently
sequencing the 16S rRNA gene using primers as described by Weisburg et al. [7]. For yeast
identification, we amplified and sequenced a fragment comprising the internal transcribed
spacer (ITS) 1, the 5.8S rRNA gene and ITS 2, along with a segment of the large subunit
rRNA gene, utilizing primers following the protocol outlined by White et al. [8].
The PCR products underwent sequencing using the Big Dye Terminator v3.1 Cycle
Sequencing Kit and were subsequently analyzed on the ABI PRISM 3100 Genetic Analyzer Sequencer (Applied Biosystems, Hitachi, Tokyo, Japan). Sequence alignment was
carried out using the BLASTN algorithm (version 2.0; National Center for Biotechnology Information) and compared against sequences from reference strains available in the
GenBank database (http://www.ncbi.nlm.nih.gov/genbank/, accessed on 3 March 2023).
Phylogenetic analyses were performed using MEGA11.
Following this initial identification, the bacterial isolates were further identified to the
species level by employing the API 20E, API STAPH, and/or API 20A systems (bioMérieux)
as per the manufacturer’s instructions, in addition to other conventional biochemical tests.
�Biol. Life Sci. Forum 2023, 31, 12
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Biol. Life Sci. Forum 2024, 31, 12
(bioMérieux) as per the manufacturer’s instructions, in addition to other conventional bi3 of 6
ochemical tests.
3. Results and Discussion
3. Results and Discussion
In this study, our initial analysis involved counting colony-forming units, which reIn this
study,
our initial
analysis
involved
colony-forming
units,
revealed
vealed
higher
bacterial
counts
in BHI
(2.51 counting
× 105 CFU/gut)
compared
to which
NA (2.25
× 105
5 CFU/gut) compared to NA (2.25 × 105 CFU/gut),
higher
bacterial
counts
in
BHI
(2.51
×
10
CFU/gut), possibly due to the greater nutrient richness in the former. Subsequently, we
possibly
to the greater
nutrient
richness
in the
Subsequently,
weanalyses,
subjected
a
subjecteddue
a selected
group of
microbial
isolates
to aformer.
comprehensive
series of
enselected
group
of
microbial
isolates
to
a
comprehensive
series
of
analyses,
encompassing
compassing gram staining, phylogenetic analysis, evaluation of biochemical properties,
gram
staining, phylogenetic
and assessment
of metabolicanalysis,
activity.evaluation of biochemical properties, and assessment
of metabolic
activity.
Gram staining yielded intriguing results, leading to the isolation of a total of eight
Gram
staining
yielded
results,
to the
of a total
of AN1eight
gram-negative
bacilli
(Figureintriguing
1): four from
NAleading
(designated
asisolation
INTA AN1-1,
INTA
gram-negative
bacilli
1): four and
fromanNA
(designated
INTA
INTA
5, INTA AN1-10,
and (Figure
INTA AN1-15)
additional
four as
from
BHIAN1-1,
(referred
to asAN1-5,
INTA
INTA
AN1-10,
and
INTA
AN1-15)
and
an
additional
four
from
BHI
(referred
to
as INTA
AC1-3, INTA AC1-6, INTA AC1-9, and INTA AC1-14). These bacilli were identified
as
AC1-3, INTA AC1-6, INTA AC1-9, and INTA AC1-14). These bacilli were identified as
dominant members of the gut microbiota in seventh stage A. diaperinus larvae. Furtherdominant members of the gut microbiota in seventh stage A. diaperinus larvae. Furthermore,
more, we observed two distinct colonies of gram-positive cocci from BHI (named INTA
we observed two distinct colonies of gram-positive cocci from BHI (named INTA AC1-4
AC1-4 and INTA AC1-8) (Figure 1). An unexpected finding in our assay was the isolation
and INTA AC1-8) (Figure 1). An unexpected finding in our assay was the isolation of two
of two yeasts from macerated intestines inoculated directly, undiluted, on BC, a medium
yeasts from macerated intestines inoculated directly, undiluted, on BC, a medium typically
typically used for the isolation of gram-positive bacteria belonging to the Bacillus cereus
used for the isolation of gram-positive bacteria belonging to the Bacillus cereus group. These
group. These yeasts stained as gram-positive, with one presenting pseudohyphae and the
yeasts stained as gram-positive, with one presenting pseudohyphae and the other not
other not (referred to as INTA AB1-1 and INTA AB1-4, respectively) (Figure 1).
(referred to as INTA AB1-1 and INTA AB1-4, respectively) (Figure 1).
◦ C.
Figure
Figure 1.
1. Pure
Pure cultures
cultures of
of the
the isolates
isolates were
were grown
grown in
in NA
NA medium
medium and
and incubated
incubated for
for 24
24 h
h at
at 29
29 °C.
Gram
× magnification
magnification immersion
Gram staining
stainingwas
was performed
performedand
andobserved
observedunder
underaa1000
1000x
immersion lens,
lens, revealing
revealing
the
following
isolates:
INTA
AN1-1
(a),
INTA
AC1-4
(b),
INTA
AC1-8
(c),
INTA
AB1-1
(d), and
the following isolates: INTA AN1-1 (a), INTA AC1-4 (b), INTA AC1-8 (c), INTA AB1-1
(d),INTA
and
INTA (e).
AB1-4
It’s important
note
thatone
only
one representative
gram-negative
is included
AB1-4
It’s(e).
important
to notetothat
only
representative
gram-negative
isolate isolate
is included
in the
in the for
figure
for clarity.
figure
clarity.
Our
thethe
genera
of of
each
of the
microbial
Our phylogenetic
phylogeneticanalysis
analysisenabled
enabledusustotodetermine
determine
genera
each
of the
microisolates
and construct
phylogenetic
trees trees
with with
the most
closely
related
species
within
each
bial isolates
and construct
phylogenetic
the most
closely
related
species
within
genus
(Figure
2). Based
on 16Son
rRNA
sequencing,
the eightthe
gram-negative
bacilli were
each genus
(Figure
2). Based
16S gene
rRNA
gene sequencing,
eight gram-negative
baclassified
Enterobacter
sp., closelysp.,
related
to related
E. hormaechei
subsp. hormaechei.
The two
cilli were as
classified
as Enterobacter
closely
to E. hormaechei
subsp. hormaechei.
gram-positive
cocci werecocci
identified
as Staphylococcus
spp., one closely
related
to S.
hominis
The two gram-positive
were identified
as Staphylococcus
spp., one
closely
related
to
subsp.
hominis
and
S.
hominis
subsp.
novobiosepticus,
and
the
other
to
S.
succinus
subsp.
casei
S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, and the other to S. succinus
and
S. succinus
succinus,
respectively
(Figure 2). (Figure 2).
subsp.
casei andsubsp.
S. succinus
subsp.
succinus, respectively
The resulting 16S rRNA gene sequences have been deposited in the GenBank database
(Table 1).
Furthermore, based on rRNA gene ITS region sequencing, we classified the two
isolated yeasts as Debaryomyces sp. and Hyphopichia sp., with the latter closely related to
Hyphopichia burtonii (Figure 3). These sequences have also been deposited in the GenBank
database (Table 1).
�l. Life Sci. Forum 2023, 31, 12
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Biol. Life Sci. Forum 2024, 31, 12
4 of 6
Figure
2. phylogenetic
The phylogenetic
16S
rRNAsequences
sequences illustrates
relationships
among
the the gra
Figure
2. The
treetree
of of
16S
rRNA
illustratesthe
the
relationships
among
gram-negative
isolates
and
type
strains
of
Enterobacter
species
(a),
as
well
as
among
the
two
gramnegative isolates and type strains of Enterobacter species (a), as well as among the two gram-posit
positive
isolates
and type
of Staphylococcus
(b). tree
The tree
constructedusing
using the
isolates
and type
strains
of strains
Staphylococcus
speciesspecies
(b). The
waswas
constructed
the neighb
neighbor-joining
method.
The
numbers
displayed
at
specific
nodes
indicate
consensus
bootstrap
joining method. The numbers displayed at specific nodes indicate consensus bootstrap values ba
values
based on 1000 replications.
on 1000
replications.
Table 1. Nucleotide lengths and GenBank accession numbers of sequences obtained from selected
gut
of Alphitobius
diaperinus.
Theisolates
resulting
16S rRNA
gene sequences have been deposited in the GenBank
base (Table 1).
Isolate
Sequenced Gene/Genes
Nucleotide Length
(bp)
da
GenBank Accession
Number
Table 1. INTA
Nucleotide
accession numbers
of sequences
obtained from selec
AN 1-1 lengths and
16SGenBank
rRNA
1401
OP339834.1
INTA
1-5
16S rRNA
1369
OP346784.1
gut isolates
ofAN
Alphitobius
diaperinus.
Isolate
INTA AN 1-1
INTA AN 1-5
INTA AN 1-10
INTA AN 1-15
INTA AC 1-3
INTA AC 1-6
INTA AC 1-9
INTA AC 1-14
INTA AC 1-4
INTA AC 1-8
INTA AB 1-1
INTA AB 1-4
INTA AN 1-10
16S rRNA
1396
INTA AN 1-15
16S rRNANucleotide Length
1397
Sequenced
Gene/Genes
INTA AC
1-3
16S rRNA
1395
(bp) 1396
INTA AC 1-6
16S rRNA
INTA
1-9
16S rRNA
16SAC
rRNA
1401 1399
INTA AC 1-14
16S rRNA
1369
16SAC
rRNA
1369 1417
INTA
1-4
16S rRNA
INTA
1-8
16S rRNA
16SAC
rRNA
1396 967
INTA AB 1-1
rRNA genes ITS region
446
16SAB
rRNA
INTA
1-4
rRNA genes ITS region 1397
612
OP346981.1
OP347118.1
GenBank
Accession Numbe
OP348220.1
OP348874.1
OP348886.1
OP339834.1
OP351273.1
OP346784.1
OP348929.1
OP348932.1
OP346981.1
OP348991.1
OP347118.1
OP348992.1
16S rRNA
1395
OP348220.1
16S
rRNAwe conducted a preliminary1396
In
addition,
species-level identification ofOP348874.1
bacteria using
API systems
and complementary biochemical 1399
tests. The isolates INTA AN1-1,
INTA AN1-5,
16S rRNA
OP348886.1
INTA AN1-10, INTA AN1-15, INTAAC1-3, INTA AC1-6, INTA AC1-9 and INTA AC1-14
16S rRNA
1369
OP351273.1
were identified as Enterobacter cloacae, INTA AC1-4 as Staphylococcus gallinarum, and INTA
rRNA
1417
OP348929.1
AC1-816S
as S.
succinus.
16S rRNA
967
OP348932.1
rRNA genes ITS region
446
OP348991.1
rRNA genes ITS region
612
OP348992.1
Furthermore, based on rRNA gene ITS region sequencing, we classified the two i
lated yeasts as Debaryomyces sp. and Hyphopichia sp., with the latter closely related to H
�Biol. Life Sci. Forum 2023, 31, 12
Biol. Life Sci. Forum 2024, 31, 12
5
5 of 6
Figure
tree tree
of rRNA
gene ITS
region
illustrates the
relationships
Figure3.3.The
Thephylogenetic
phylogenetic
of rRNA
gene
ITSsequences
region sequences
illustrates
the relations
among
the
yeast
isolates
and
the
type
strains
of
Debaryomyces
and
Hyphopichia
species.
The
tree
was The tree
among the yeast isolates and the type strains of Debaryomyces and Hyphopichia species.
constructed
using
the
neighbor-joining
method,
and
the
numbers
shown
at
specific
nodes
represent
constructed using the neighbor-joining method, and the numbers shown at specific nodes repre
consensus
values
derived
from 1000
consensusbootstrap
bootstrap
values
derived
fromreplications.
1000 replications.
Our findings diverged from previous reports in recent years on the bacterial and fungal
In addition,
wepolystyrene
conducted
a A.
preliminary
species-level
identification
of bacteria
u
diversity
in the gut of
fed
diaperinus population
[4]. These
discrepancies
in
API systems and
complementary
biochemical
tests.
Thediet.
isolates
INTA
AN1-1, INTA A
microorganism
genera
may be attributed
to differences
in larval
While
our laboratory
breeding
on feed
for baby
chicks,INTAAC1-3,
A. diaperinus colonies
other studies
exposed
5, INTArelies
AN1-10,
INTA
AN1-15,
INTA in
AC1-6,
INTAwere
AC1-9
and INTA A
to
plastic
compounds
and
microorganisms
potentially
involved
in
plastic
degradation.
14 were identified as Enterobacter cloacae, INTA AC1-4 as Staphylococcus gallinarum,
A separate Italian master’s thesis on the microbiological aspects and chemical comINTA
AC1-8 as S. succinus.
position of A. diaperinus, Tenebrio molitor, and Zophobas morio larvae intended for human
Our findings
diverged
from previous
in recent
years on the bacterial
and
consumption
supported
our findings
regarding reports
the presence
of Enterobacteriaceae
and
gal diversityspp.
in the
guttotal
of polystyrene
fed A. diaperinus
[4]. and
These
discrepan
Staphylococcus
in the
mesophilic bacterial
load, along population
with enterococci
lactic
in microorganism
genera
may5 be
to[9].
differences in larval diet. While our la
acid
bacteria, all ranging
between
andattributed
7 log CFU/g
Furthermore,
microbial
an industrial
productioncolonies
cycle of A.indiaperinus
atory
breeding relies
on dynamics
feed for during
baby chicks,
A. diaperinus
other studies w
intended
for
human
consumption
were
characterized
[10].
While
bacterial
diversity
exposed to plastic compounds and microorganisms potentially involved indeplastic de
creased during rearing, the number of aerobic endospores remained at 4.0 log CFU/g.
dation.
Coagulase-positive staphylococci were not detected, but fungal isolates from the genera
A separate
Italian
master’s
thesis on the microbiological aspects and chemical c
Aspergillus
and Fusarium
were
recovered.
position
of A.underscores
diaperinus, the
Tenebrio
molitor,
andofZophobas
morio larvaeprocedures,
intended for hu
Our study
potential
influence
rearing environments,
hygiene
measures,
and insectour
feedfindings
on insect microbiota.
consumption
supported
regarding the presence of Enterobacteriaceae
The disparities
between
analysis
and phenotypic
data for
certain
isolates and l
Staphylococcus
spp.
in thephylogenetic
total mesophilic
bacterial
load, along
with
enterococci
suggest
the possible
existence between
of new species
or 7subspecies.
Further
acid bacteria,
all ranging
5 and
log CFU/g
[9]. comprehensive studies,
including the sequencing of entire genomes for the three kinds of bacteria isolated from the
Furthermore, microbial dynamics during an industrial production cycle of A. dia
gut microbiota of A. diaperinus, are warranted. Bioinformatics analysis currently underway
inusprovide
intended
for insights
humaninto
consumption
were
characterized
[10]. While
bacterial dive
will
deeper
this ecological
niche,
potentially enhancing
insecticidal
decreased
during
rearing,
the number
of aerobic
strategies
against
beetles
with significant
poultry
impact. endospores remained at 4.0 log CF
In conclusion, thisstaphylococci
study sheds light
on not
the diverse
microbiota
of A.isolates
diaperinus,
re- the ge
Coagulase-positive
were
detected,
but fungal
from
vealing
potential
implications
for
insect
pest
management
and
offering
avenues
for
future
Aspergillus and Fusarium were recovered.
research into insect-microbe interactions.
Our study underscores the potential influence of rearing environments, procedu
hygiene measures, and insect feed on insect microbiota.
The disparities between phylogenetic analysis and phenotypic data for certain
lates suggest the possible existence of new species or subspecies. Further comprehen
studies, including the sequencing of entire genomes for the three kinds of bacteria isol
�Biol. Life Sci. Forum 2024, 31, 12
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Author Contributions: Conceptualization, G.I.A. and D.H.S.; methodology, G.I.A. and D.H.S.;
software, G.I.A. and D.H.S.; validation, G.I.A. and D.H.S.; formal analysis, G.I.A. and D.H.S.; investigation, G.I.A. and D.H.S.; resources, D.H.S.; data curation, G.I.A. and D.H.S.; writing—original draft
preparation, G.I.A. and D.H.S.; writing—review and editing, D.H.S.; visualization, G.I.A. and D.H.S.;
supervision, D.H.S.; project administration, D.H.S.; funding acquisition, D.H.S. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by INTA 2023-PD-L06-I116.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Kureˇcka, M.; Kulma, M.; Petˇríˇcková, D.; Plachy, V.; Kouˇrimská, L. Larvae and pupae of Alphitobius diaperinus as promising protein
alternatives. Eur. Food Res. Technol. 2021, 247, 2527–2532. [CrossRef]
Rumbos, C.I.; Karapanagiotidis, I.T.; Mente, E.; Athanassiou, C.G. The lesser mealworm Alphitobius diaperinus: A noxious pest or
a promising nutrient source? Rev. Aquacult. 2019, 11, 1418–1437. [CrossRef]
Cucini, C.; Funari, R.; Mercati, D.; Nardi, F.; Carapelli, A.; Marri, L. Polystyrene shaping effect on the enriched bacterial community
from the plastic-eating Alphitobius diaperinus (Insecta: Coleoptera). Symbiosis 2022, 86, 305–313. [CrossRef]
Cucini, C.; Leo, C.; Vitale, M.; Frati, F.; Carapelli, A.; Nardi, F. Bacterial and fungal diversity in the gut of polystyrene-fed
Alphitobius diaperinus (Insecta: Coleoptera). Animal Gene 2020, 17–18, 200109. [CrossRef]
Vaughan, J.A.; Turner, E.C.; Ruszler, P.L. Infestation and Damage of Poultry House Insulation by the Lesser Mealworm, Alphitobius
diaperinus (Panzer). Poult. Sci. 1984, 63, 1094–1100. [CrossRef]
Lu, F.; Kang, X.; Jiang, C.; Lou, B.; Jiang, M.; Way, M. Isolation and characterization of bacteria from midgut of the rice water
weevil (Coleoptera: Curculionidae). Environ. Entomol. 2013, 42, 874–881. [CrossRef] [PubMed]
Weisburg, W.G.; Barns, S.M.; Pelletier, D.A.; Lane, D.J. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 1991,
173, 697–703. [CrossRef] [PubMed]
White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics. In
PCR Protocols: A Guide to Methods and Applications; Academic Press: New York, NY, USA, 1990; pp. 315–322.
Martinis, V. Aspetti Microbiologici e Composizione Chimica di larve di Alphitobius diaperinus, Tenebrio molitor e Zophobas morio
Destinati al Consumo Umano. Master’s Thesis, Università di Pisa, Pisa, Italy, 2020.
Wynants, E.; Crauwels, S.; Verreth, C.; Gianotten, N.; Lievens, B.; Claes, J.; Van Campenhout, L. Microbial dynamics during
production of lesser mealworms (Alphitobius diaperinus) for human consumption at industrial scale. Food Microbiol. 2018, 70,
181–191. [CrossRef] [PubMed]
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Isolation and identification of culturable gut microbiota in the larval stage of
lesser mealworm (Alphitobius diaperinus)
Gisele Ivonne Antonuccio*1,2 and Diego Herman Sauka 1,3
1
Instituto Nacional de Tecnología Agropecuaria (INTA), Instituto de Microbiología y Zoología Agrícola (IMYZA), Hurlingham, Buenos Aires, Argentina
2
Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), Buenos Aires, Argentina
3
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
*Correspondence: antonuccio.gisele@inta.gob.ar
INTRODUCTION
The highly prevalent pest Alphitobius diaperinus (Coleoptera:
Tenebrionidae) causes significant structural damage in poultry
farms. Despite previous investigations on its carriage of
pathogenic microorganisms, our understanding of its microbiome
remains limited. This study aimed to analyze the diversity of
culturable gut microbiota in A. diaperinus obtained from
laboratory breeding.
MATERIALS AND METHODS
Fifteen seventh instar larvae underwent a 24-hour starvation
period, followed by surface disinfection. Dissected midguts were
homogenized and plated on nutrient agar (NA), brain heart
infusion agar (BHI), and Bacillus cereus agar (BC). The cultured
isolates were subjected to gram staining, phylogenetic analysis,
biochemical property evaluation, and metabolic activity
assessment.
RESULTS AND DISCUSSION
-Higher bacterial counts in BHI (2.51x105 CFU/gut) compared to
NA (2.25x105 CFU/gut), possibly due to nutrient richness.
-NA exhibited a dominant colony morphology of gram-negative
bacilli, while BHI displayed additional distinct colonies of grampositive cocci. Surprisingly, yeast-like colonies were observed on
BC plates (Figure 1).
-Based on 16S rRNA gene sequences, eight bacterial isolates were
identified as Enterobacter sp., and two as Staphylococcus sp. Using
RNA gene ITS region sequences, two yeast isolates were
identified as Debaryomyces sp. and Hyphopichia sp.
-The resulting 16S rRNA gene sequences have been deposited in
the GenBank database (Table 1). Based on rRNA gene ITS region
sequencing, we classified the two isolated yeasts. These sequences
have also been deposited in the GenBank database (Table 1).
-A preliminary species-level identification of bacteria (Enterobacter
cloacae, Staphylococcus gallinarum, and Staphylococcus succinus) was
achieved using API systems and complementary biochemical
tests.
-Discrepancies between phylogenetic analysis (Figures 2 and 3)
and phenotypic data suggest the potential existence of new
species or subspecies. Further comprehensive studies are required
to confirm this hypothesis.
Figure 1. Pure cultures of the isolates were grown in NA medium and incubated for 24 hours at 29°C. Gram staining was performed and observed under a 1000x
magnification immersion lens, revealing the following isolates: INTA AN1-1 (a), INTA AC1-4 (b), INTA AC1-8 (c), INTA AB1-1 (d), and INTA AB1-4 (e). It's important
to note that only one representative gram-negative isolate is included in the figure for clarity.
Figure 2. The phylogenetic tree of 16S rRNA sequences illustrates the relationships among the gram-negative isolates and type strains of Enterobacter species (a), as
well as among the two gram-positive isolates and type strains of Staphylococcus species (b). The tree was constructed using the neighbor-joining method. The numbers
displayed at specific nodes indicate consensus bootstrap values based on 1,000 replications.
Table 1. Nucleotide lengths and GenBank accession numbers of sequences obtained from
selected gut isolates of Alphitobius diaperinus.
Isolate
Sequenced gene/genes
Nucleotide
length (bp)
GenBank accession
number
INTA AN 1-1
16S rRNA
1401
OP339834.1
INTA AN 1-5
16S rRNA
1369
OP346784.1
INTA AN 1-10
16S rRNA
1396
OP346981.1
INTA AN 1-15
16S rRNA
1397
OP347118.1
INTA AC 1-3
16S rRNA
1395
OP348220.1
INTA AC 1-6
16S rRNA
1396
OP348874.1
INTA AC 1-9
16S rRNA
1399
OP348886.1
INTA AC 1-14
16S rRNA
1369
OP351273.1
INTA AC 1-4
16S rRNA
1417
OP348929.1
INTA AC 1-8
16S rRNA
967
OP348932.1
INTA AB 1-1
rRNA genes ITS region
446
OP348991.1
INTA AB 1-4
rRNA genes ITS region
612
OP348992.1
1
Figure 3. The phylogenetic tree of rRNA gene ITS region sequences illustrates the
relationships among the yeast isolates and the type strains of Debaryomyces and
Hyphopichia species. The tree was constructed using the neighbor-joining method, and
the numbers shown at specific nodes represent consensus bootstrap values derived
from 1,000 replications.
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Isolation and identification of culturable gut microbiota in the larval stage of lesser mealworm <em>(Alphitobius diaperinus</em>)
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Antonuccio, Gisele
Sauka, Diego
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Publicado en Biology and Life Sciences Forum 2024, 31, 12.
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2024
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En
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B.S.0184 - B.S.0184-1
Abstract
A summary of the resource.
La plaga altamente prevalente <em>Alphitobius diaperinus</em> (Coleoptera: Tenebrionidae) causa daño estructural significativo en granjas avícolas. A pesar de investigaciones previas sobre su transporte de microorganismos patógenos, nuestro conocimiento de su microbioma sigue siendo limitado. Este estudio tuvo como objetivo analizar la diversidad de la microbiota intestinal cultivable en <em>A. diaperinus</em> obtenida de la cría de laboratorio.<br />Trabajo presentado en la Segunda Conferencia Electrónica Internacional de Microbiología (2nd International Electronic Conference on Microbiology), del 1 al 15 de Dicimbre de 2023.
Alphitobius diaperinus
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SECUENCIACIÓN MASIVA DE GENOMAS BACTERIANOS ASOCIADOS A
Alphitobius diaperinus (COLEOPTERA: TENEBRIONIDAE)
Antonuccio, Gisele 1,2*; Sauka, Diego 1,3
(1) Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola (IMYZA). Hurlingham. Buenos Aires. Argentina; (2) Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), Buenos Aires,
Argentina; (3) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Buenos Aires. Argentina.
* antonuccio.gisele@inta.gob.ar
INTRODUCCIÓN
Alphitobius diaperinus (Coleoptera: Tenebrionidae),
conocido como el escarabajo de la cama de pollos, es
una plaga de origen africano presente en Argentina y
de gran importancia en el sector avícola.
Investigaciones previas, mediante el análisis de ciertos
caracteres fenotípicos y filogenéticos del gen rRNA
16S, han llevado a la identificación de tres bacterias
que componen la microbiota intestinal cultivable de las
larvas de este insecto. Se identificaron ocho bacilos
Gram negativos dominantes como Enterobacter sp. y
dos diferentes cocos Gram positivos como
Staphylococcus sp.
OBJETIVO
Secuenciar los genomas completos de una de las cepas
de Enterobacter sp., denominada INTA AN1-1, así como
de las dos cepas de Staphylococcus sp., denominadas
INTA AC1-4 e INTA AC1-8, con el fin de investigar la
posibilidad de que representen nuevas especies o
subespecies bacterianas.
Tabla 1. Principales características de los ensamblados de los genomas borradores de las cepas INTA AN1-1, INTA AC1-4 e INTA AC1-8
obtenidos usando QUAST v4.4 y CheckM v1.0.18.
Cantidad total de contigs
Contig más grande (número
de nucleótidos)
Largo total (número de
nucleótidos)
Contenido GC (%)
Valor N50
Valor N75
Valor L50
Valor L75
Completitud del ensamblaje
(%)
Presunta contaminación (%)
INTA AN1-1
INTA AC1-4
INTA AC1-8
75
135
84988
51
206092
2238227
2728651
31.35
32789
17154
21
45
99.31
32.88
88384
61408
10
19
99.45
0.55
1.93
621970
5083490
55
210427
111550
7
15
99.73
- Los valores obtenidos superan los umbrales de corte de ANI del
96% y DDH del 79% establecidos para que las bacterias en estudio
sean consideradas nuevas especies o subespecies (Tabla 2) .
- Estas bacterias se han encontrado en un nuevo nicho ecológico,
diferente al de su descripción original: masa fermentada en China,
sangre humana en Estados Unidos e inclusiones de plantas y
suelos en ámbar dominicano con entre 25 y 35 millones de años
de antigüedad, respectivamente.
Tabla 2. Cepas de especies tipo más estrechamente relacionadas con INTA AN1-1 (azul), INTA AC1-4 (verde) e INTA AC1-8 (amarillo),
respectivamente, con sus valores de hibridación DNA–DNA digital (dDDH) y diferencia porcentual en el contenido de G+C utilizando
el pipeline TYGS, valores de ANI (Average Nucleotide Identity) y Tetra z-score.
Cepa
N° ensamblado
GenBank
Enterobacter hormaechei
subsp. xiangfangensis
GCF_001729785
LMG 27195
MATERIALES Y MÉTODOS
Enterobacter hormaechei
subsp. oharae DSM 16687 GCF_001729705
Enterobacter hormaechei
subsp. steigerwaltii DSM GCF_001729725
16691
Extracción de DNA
genómico total de
cada cepa por kit
Secuenciación genómica
por Illumina NovaSeq 6000
Ensamblado de las lecturas + análisis
bioinformáticos con kbase.us,
usegalaxy.org, tygs.dsmz.de y
jspecies.ribohost.com.
RESULTADOS Y DISCUSIÓN
- Las principales características de los ensamblados de
los genomas borradores de las cepas INTA AN1-1, INTA
AC1-4 e INTA AC1-8 se presentan en la tabla 1.
- Las cepas de especies tipo más estrechamente
relacionadas con INTA AN1-1, INTA AC1-4 e INTA AC1-8
fueron aquellas pertenecientes a Enterobacter
hormaechei subsp. xiangfangensis, Staphylococcus
hominis subsp. novobiosepticus y Staphylococcus
succinus subsp. succinus, respectivamente (Tabla 2).
0.19
Diferencia
dDDH (d0, dDDH (d4, dDDH (d6,
contenido
en %)
en %)
en %)
G+C (en %)
ANIb [%]
ANIm [%]
Tetra zscore
83.9
93.0
88.2
0.28
98.81
99.19
0.99915
78.6
76.2
81.0
0.58
96.96
97.28
0.99875
75.6
75.8
78.4
0.55
96.72
97.24
0.99878
Staphylococcus hominis
subsp. novobiosepticus
CCUG 42399
GCF_002902465
84.7
92.4
88.9
0.04
98.96
99.15
0.99852
Staphylococcus hominis
NCTC 11320
GCF_002901845
88.2
79.4
89.5
0.03
97.39
97.73
0.99944
Staphylococcus petrasii
subsp. jettensis CCUG
62657
GCF_002902105
27.5
22.9
25.4
1.9
79.48
85.27
0.94967
Staphylococcus succinus
GCA_001006765
DSM 14617
92.0
82.1
92.9
0.06
97.79
97.95
0.99883
Staphylococcus casei DSM
GCF_002902445
15096
87.4
65.0
86.1
0.16
95.43
95.76
0.99717
Staphylococcus equorum
GCA_900458565
NCTC 12414
33.1
23.4
29.7
0.23
79.24
84.39
0.99105
CONCLUSIÓN
- Este estudio contribuye a una mejor comprensión de la biología
de la microbiota intestinal del escarabajo de la cama de pollos,
resaltando la adaptación de estas bacterias a su nuevo entorno.
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Trabajos, Tesis, Tesinas
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Secuenciación masiva de genomas bacterianos asociados a <em>Alphitobius diaperinus</em> (Coleoptera: Tenebrionidae)
Creator
An entity primarily responsible for making the resource
Antonuccio, Gisele
Sauka, Diego
Date
A point or period of time associated with an event in the lifecycle of the resource
2024
Language
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Es
Type
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Póster académico
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B.S.0185
Abstract
A summary of the resource.
Trabajo presentado en el XVI Congreso Argentino de Microbiología (CAM 2024), 21 al 23 de agosto de 2024, Ciudad Autonoma de Buenos Aires. Este estudio contribuye a una mejor comprensión de la biología de la microbiota intestinal del escarabajo de la cama de pollos, resaltando la adaptación de estas bacterias a su nuevo entorno.
Alphitobius diaperinus
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Article
Initial Sublethal Exposure to an Argentine Bacillus thuringiensis
Strain Induces Chronic Toxicity and Delayed Mortality in
Alphitobius diaperinus (Coleoptera: Tenebrionidae)
Gisele Ivonne Antonuccio 1,2, * , Lucas Candás 1
1
2
3
*
and Diego Herman Sauka 1,3
Instituto Nacional de Tecnología Agropecuaria (INTA), Instituto de Microbiología y Zoología
Agrícola (IMYZA), Buenos Aires B1686IGC, Argentina; candas.lucas@inta.gob.ar (L.C.);
sauka.diego@inta.gob.ar (D.H.S.)
Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), Buenos Aires C1107ADR, Argentina
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires C1425FQB, Argentina
Correspondence: antonuccio.gisele@inta.gob.ar
Simple Summary
Pest control in agriculture and livestock is a constant challenge, particularly when insect
pests affect animal production systems. Although agrochemicals have traditionally been
the main control strategy, environmentally friendly alternatives are increasingly needed.
Bacillus thuringiensis is a widely used bacterium for insect control that acts when ingested
and is valued for its safety and target specificity. However, the initial effects of concentrations that do not immediately kill insects but weaken them over time have been little studied
in beetle pests. In this work, we evaluated the initial sublethal effects of an Argentine
B. thuringiensis strain on Alphitobius diaperinus larvae after 14 days of dietary exposure and
followed the insects throughout their life cycle to assess chronic toxicity. Larvae exposed to
the bacterium showed significant reductions in weight and body size, altered nutritional
reserves, and reduced survival compared with untreated individuals. Even insects that
initially survived exhibited significant delayed mortality, indicating long-term irreversible
damage. These results demonstrate that B. thuringiensis can reduce beetle populations not
only by killing insects directly but also by weakening them through chronic effects, supporting its use as an effective and sustainable biotechnological tool for pest management.
Abstract
Academic Editor: Nickolas
G. Kavallieratos
Received: 26 December 2025
Revised: 8 February 2026
Accepted: 13 February 2026
Published: 18 February 2026
Copyright: © 2026 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license.
Insects 2026, 17, 213
Bacillus thuringiensis is the most extensively studied entomopathogenic bacterium worldwide; however, its sublethal effects on beetles remain poorly characterized. The aim of this
study was to evaluate the toxicity of a previously selected Argentine strain of B. thuringiensis
on second-instar Alphitobius diaperinus larvae during an initial 14 days of exposure, and to
assess its effects at day 14 and throughout the remainder of the life cycle until death. Three
treatments were applied: control, LC30 , and LC50 . Larval, pupal, and adult weight and body
surface area were recorded, and nutritional composition was quantified using colorimetric
methods. Insect status was monitored every 48–72 h over a total period of 540 days, until
the death of the last individual. Among the evaluated variables, statistically significant
differences between control and treatment groups were detected in larval area and weight,
in the survival analysis and in two nutritional components: total protein and lipid content
per larva. Overall, the results demonstrate that initial sublethal exposure to B. thuringiensis
induces chronic lethal effects with delayed mortality in A. diaperinus, indicating irreversible
physiological damage. This provides valuable information not only for understanding
the biology of this insect but also for stakeholders involved in the productive scaling of
beetle-targeted bioinputs.
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Keywords: Alphitobius diaperinus; Bacillus thuringiensis; sublethal effects; biocontrol; virulence
1. Introduction
The lesser mealworm, Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae) [1], is
a key pest in poultry production systems. In addition to causing direct damage to facilities,
it is recognized as a potential vector of avian pathogens, including bacteria, viruses, and
parasites, posing significant sanitary risks and potentially leading to substantial economic
losses [2]. The intensive use of chemical insecticides for its control has resulted in resistant
populations, as well as concerns about residues in poultry products and risks to animal
and human health. These challenges underscore the need for alternative, environmentally
safe control strategies [3,4].
Among microbial control agents, Bacillus thuringiensis is the most widely used entomopathogenic bacterium. During sporulation, it produces parasporal crystalline inclusions
composed of proteins (Cry and Cyt) that exhibit selective toxicity against insect larvae
upon ingestion. This bacterium can also secrete pesticidal proteins during the vegetative
stage (Vpa/Vpb and Vip). To date, hundreds of B. thuringiensis pesticidal proteins have
been described [5], some of which have been developed into bioinsecticide formulations
or expressed in transgenic crops. Their activity spans multiple insect orders, including
Lepidoptera, Diptera, Coleoptera, and Hemiptera.
The first B. thuringiensis strain with activity against coleopterans was reported by
Krieg et al., 1983 [6]. Since then, several pesticidal proteins have been associated with
toxicity to A. diaperinus larvae [7,8]. Nevertheless, compared with lepidopteran pests,
the market for coleopteran-targeting bioinsecticides remains less developed, partly due
to the historical focus on caterpillars, the limited availability and narrower activity of
coleopteran-active B. thuringiensis toxins, and the cryptic feeding habits of many beetle
larvae, which reduce their exposure to B. thuringiensis and complicate effective application.
Recent evaluations of local B. thuringiensis strains have identified promising candidates
for controlling A. diaperinus. Pérez et al., 2025 [9] selected INTA Mo4-4 as highly toxic
to larvae, demonstrating that its insecticidal activity is predominantly associated with
the spore–crystal pellet, consistent with the involvement of Cry proteins. In that study,
INTA Mo4-4 showed the highest toxicity among 41 evaluated strains and caused mortality
levels 2.7-fold higher than those of the reference strain B. thuringiensis svar. morrisoni
tenebrionis DSM 2803. Previous reports have shown that such parasporal crystal proteins—
including Cry3Aa, Cry3Bb, Cry8Ca and proteins with dual activity against Diptera and
some coleopterans, including Cry4B, Cry10, Cry11A and Cyt1A—constitute the main
virulence factors of B. thuringiensis against A. diaperinus [7,8].
However, most studies have focused on acute toxicity, while the potential sublethal
effects of B. thuringiensis exposure remain largely unexplored [10–13]. This is particularly relevant because, under field or farm conditions, environmental factors such as UV
light, rainfall and microbial degradation often reduce the persistence and availability of
B. thuringiensis toxins [14]. As a result, insects may be exposed to initial sublethal doses
that, although insufficient to cause immediate mortality during the early stages of exposure,
could affect development, reproduction, and overall fitness. To date, studies addressing
such sublethal effects in A. diaperinus are scarce. Understanding how initial sublethal concentrations of B. thuringiensis impact this pest could provide valuable insights for integrated
pest management and contribute to more sustainable control strategies. Therefore, the
objective of this work was to evaluate the initial sublethal effects and subsequent chronic
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toxicity of an Argentine B. thuringiensis strain on the development, survival and fitness of
A. diaperinus.
2. Materials and Methods
2.1. Production of Bacillus thuringiensis INTA Mo4-4 Active Ingredient
INTA Mo4-4 is an Argentine B. thuringiensis strain isolated from stored-product dust
collected in the locality of Chacabuco, Buenos Aires Province, Argentina. The strain is
preserved in the Bacterial Collection of the Instituto de Microbiología y Zoología Agrícola,
Instituto Nacional de Tecnología Agropecuaria (IMYZA-INTA), as previously reported by
Pérez et al. (2025) [9]. Bacillus thuringiensis INTA Mo4-4 biomass was produced following
the protocol of Pérez (2017) [15] with minor modifications. An optimized BM broth (containing 2.5 g NaCl, 1 g KH2 PO4 , 2.5 g K2 HPO4 , 0.25 g MgSO4 ·7H2 O, 0.1 g MnSO4 ·H2 O, 5 g
glucose and 6 g yeast extract per liter of distilled water, adjusted to pH 7.2) was prepared
and divided into 12 Erlenmeyer flasks (50 mL per flask). Each flask was inoculated with
50 µL of a highly concentrated stock suspension of the spore-crystal complex. Cultures
were incubated in the dark at 28 ◦ C with shaking (250 rpm) for 72 h, until autolysis occurred. The biomass (spore-crystal complex) was collected by centrifugation (10,000 g, 4 ◦ C,
20 min), washed three times with sterile distilled water, dried at 28 ◦ C for four days, and
ground to a fine powder that was stored at −20 ◦ C until further use in subsequent analyses
and bioassays.
2.2. Bioassays for Toxicity
Biological tests with a spore-crystal complex suspension were conducted, except that
a series of six concentrations (concentration range: 37.13–320 µg/mL; dilution factor: 0.65)
were prepared to establish the concentration-response relationship by Probit analysis. Fortyeight larvae (24 larvae per plate) were tested for each concentration and bioassay date.
Bioassays were performed against second instar larvae of A. diaperinus using the diet
incorporation method previously described Pérez (2017) [15]. Artificial larval diet was
prepared daily (133.3 g chicken feed, 10 g agar, 1 L deionized water) and sterilized (121 ◦ C,
15 min). Preservatives (ascorbic acid 2.5 g/L, sorbic acid 1.25 g/L, nipagin 2.08 g/L) were
added after cooling to 55 ◦ C. Strain suspensions were incorporated into freshly prepared
diet made on the same day, based on chick starter feed (4 mL per 36 mL diet per Falcon
tube), and 400 µL of diet were dispensed per well in 24-well plates. Second-instar larvae
were individually placed in wells. Mortality was recorded after 14 days at 29 ◦ C. Four
independent bioassays fulfilling the statistical criteria for B. thuringiensis were chosen as
described by Iriarte & Caballero 2001 [16], and LC30 and LC50 values were estimated using
Probit analysis [17] in IBM SPSS Statistics v19. To ensure robustness and reproducibility,
only bioassays showing coefficients of variation ≤ 20% were considered valid.
Two series of bioassays were performed: first, six concentrations of spores and crystals
were tested for acute toxicity; second, sublethal bioassays using LC30 and LC50 concentrations were conducted on surviving larvae to assess effects on development and fitness.
The selection of LC30 and LC50 was based on bibliographic references that revealed
sublethal effects of B. thuringiensis on pest insect larvae, both individually and in combination with other control strategies [18,19].
2.3. Evaluation of Sublethal and Chronic Effects
Initial sublethal effects were evaluated after 14 days of exposure to the wet diet (LC30
and LC50 , as defined in Section 2.2). To quantify growth inhibition, larvae were weighed
in pools of 48 individuals and photographed in groups of four to estimate their body area
using Image J software (version 1.54g; National Institutes of Health, Bethesda, MD, USA).
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Following this initial exposure phase, individuals were transferred to and maintained in
separate test tubes with a dry (untreated) diet to prevent cannibalism and ensure precise
individual tracking, while allowing for the assessment of delayed mortality. A. diaperinus
specimens were monitored throughout their entire life cycle. Survival, molting, pupation,
and adult emergence were recorded every 48–72 h. Upon reaching these stages, pupae and
adults were weighed and photographed. Sex determination at the pupal stage followed
the morphological criteria of Esquivel et al., 2012 [20].
To identify chronic toxicity and determine the maximum life expectancy for A. diaperinus
per treatment, monitoring was extended for up to 540 days from hatching. This period was
established based on the maximum lifespan recorded in the laboratory conditions, where
0.42% of the control, 0.46% of the LC30 , and 0.55% of the LC50 populations reached this age.
This extended observation window is essential to capture physiological “hidden costs” and
delayed mortality—which define here as chronic lethal effects—that standard short-term
bioassays typically overlook, providing a comprehensive view of the long-term impact of
B. thuringiensis exposure.
Survival analysis was conducted using Kaplan-Meier curves, and statistical differences
among treatments were assessed using the log-rank (Mantel-Cox) test applying a Bonferroni
correction for multiple comparisons. To evaluate the rate of mortality within each treatment,
lethal time (LT50 and LT90 ) values were estimated using bootstrap confidence intervals [21].
2.4. Biochemical Analyses of Surviving Larvae
Surviving larvae from control, LC30 , and LC50 treatments were randomly pooled and
sacrificed for biochemical assays to evaluate the impact of Bt exposure on energy reserves.
Proteins: Following the protocol of Brogdon 1984 [22], samples (pools of 2–14 larvae,
depending on size) were homogenized in phosphate-buffered saline (PBS, pH 7.4) and
centrifuged at 15,400× g for 3 min at 4 ◦ C to separate the supernatant from the pellet. The
resulting supernatants were analyzed using Bradford reagent in 96-well plates. Protein
concentrations were calculated from bovine serum albumin (BSA) standard curves and
normalized to larval biomass (µg protein/mg larval weight) by measuring absorbance at
595 nm using a microplate reader.
Lipids: Total lipids were extracted as described by Anschau et al., 2017 [23] with slight
modifications. Pools of ~10 larvae were homogenized in chloroform:methanol (2:1, v/v)
mixture. After centrifugation (15,400× g for 3 min at 4 ◦ C) to separate the supernatant from
the pellet, supernatants were reacted with concentrated H2 SO4 and vanillin-phosphoric
acid reagent for colorimetric quantification. Absorbance was recorded at 530 nm, and lipid
content was expressed as µg/mg of larval weight.
Sugars and glycogen: According to Yuval et al., 1998 [24] sugars were extracted using
a chloroform:methanol (1:2, v/v) solution, whereas glycogen was obtained by aqueous
extraction from the resulting pellets in a subsequent step. Both analytes were reacted with
anthrone in H2 SO4 using different reagent proportions following the reference method,
and absorbance was measured at the same wavelength (625 nm). Sugars were quantified
using glucose-based standard curves, while glycogen standards (Fermentas, molecular
biology grade, 20 mg mL−1 ) were used for glycogen determination.
For all variables analyzed in this study, including biological parameters and biochemical assays, statistical assumptions (normality via Shapiro-Wilk and homogeneity of
variance via Levene’s test) were verified to ensure the appropriateness of the statistical tests.
Differences among treatments were analyzed using one-way analysis of variance (ANOVA)
for parametric data or the Kruskal–Wallis test for non-parametric data, with Bonferroni
corrections applied where appropriate.
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3. Results
3.1. Lethal and Sublethal Concentration Estimates
Concentration–mortality responses for the four bioassays are summarized in Table 1.
LC30 values ranged from 61.55 to 84.34 µg/mL, and LC50 values ranged from 117.83 to
154.55 µg/mL. The slopes of the probit regressions varied between 1.42 and 2.18. χ2 values
(4 df) indicated a satisfactory model fit across all assays. Coefficients of variation were below
20% for both calculated LC levels. The mean LC30 (68.91 µg/mL) and LC50 (135.74 µg/mL)
were selected as the initial sublethal exposure levels for subsequent experiments.
Table 1. Sublethal concentrations of a spore-crystal suspension of B. thuringiensis INTA Mo4-4 against
second-instar larvae of A. diaperinus 14 days post treatment.
Assay
1
2
3
4
Mean
CV 2
LC30 1 (µg/mL)
LC50 1 (µg/mL)
67.76
[52.64–81.64]
84.34
[65.65–102.24]
61.98
[40.35–81.21]
61.55
[19.66–95.36]
117.83
[99.24–140.86]
154.55
[128.08–194.19]
144.87
[113.21–198.76]
125.69
[77.50–229.18]
68.91
15.49%
135.74
12.46%
Slope 3
χ2 (4 df) 4
2.18
3.97
1.99
1.85
1.42
4.58
1.69
7.88
LC30 1 and LC50 1 (lethal concentration) average of four repetitions + 95% confidence limits for concentration;
2 coefficient of variation; 3 slope; 4 χ2 with four degrees of freedom (df).
3.2. Effects on Larval Performance and Development
The impact of initial sublethal concentrations (LC30 and LC50 ) of the spore-crystal
suspension of INTA Mo4-4 on selected biological parameters of A. diaperinus is summarized
in Table 2. Additional data are provided in the Supplementary Information, including
Table S1 (Individual larval weight), Table S2 (Individual larval area), Table S3 (Individual
larval and pupal stage duration), and Table S4 (Pupae and adult area and weight).
Table 2. Sublethal effects of LC30 and LC50 on biological parameters of A. diaperinus. The average,
minimum, and maximum values ± standard error (S.E.) are indicated for each parameter.
Variable
Larval weight (mg)
Larval area (mm2 )
Larval stage
duration from
hatching (days)
Larval stage
duration since the
end of Bt intake
(days)
Pupation rate (%)
Pupal stage
duration (days)
Control (Mean ±
S.E. [min–max])
0.37 B ± 0.02
[0.28–0.42]
1.36 B ± 0.08
[1.17–1.67]
n
192
192
LC30 (Mean ± S.E.
[min–max])
0.25 A ± 0.02
[0.23–0.28]
0.95 A ± 0.08
[0.87–1.06]
n
180
180
LC50 (Mean ± S.E.
[min–max])
0.20 A ± 0.02
[0.17–0.22]
0.82 A ± 0.08
[0.71–0.95]
n
178
178
94.69 A ± 9.17
[78.31–105.00]
106
101.13 A ± 10.59
[80.92–114.48]
71
100.65 A ± 12.97
[78.70–122.60]
28
76.69 A ± 9.17
[60.31–87.00]
106
83.13 A ± 10.59
[62.92–96.48]
71
82.65 A ± 12.97
[60.70–104.60]
28
42.79 A ± 15.17
[4.17–81.25]
5.71 A ± 0.30
[5.00–6.36]
106
95
26.58 A ± 15.17
[0.00–54.55]
6.35 A ± 0.35
[5.93–6.62]
71
65
11.78 A ± 15.17
[0.00–32.39]
6.95 A ± 0.43
[6.29–7.60]
28
26
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Table 2. Cont.
Variable
Pupal area (mm2 )
Pupal weight (mg)
Adults rate (%)
Adult area (mm2 )
Adult weight (mg)
Control (Mean ±
S.E. [min–max])
10.21 A ± 1.27
[9.32–11.10]
11.30 A ± 1.74
[10.10–12.50]
42.79 A ± 15.11
[4.17–81.25]
9.87 A ± 0.92
[8.70–11.28]
9.48 A ± 1.13
[8.42–11.04]
n
87
87
106
80
80
LC30 (Mean ± S.E.
[min–max])
9.78 A ± 1.04
[8.44–11.21]
10.95 A ± 1.42
[8.75–13.05]
26.29 A ± 15.11
[0.00–53.41]
9.72 A ± 0.92
[8.54–11.38]
9.65 A ± 1.13
[7.65–11.35]
LC50 (Mean ± S.E.
[min–max])
n
9.74 A ± 1.27
[7.80–11.68]
10.58 A ± 1.74
[8.12–13.04]
11.78 A ± 15.11
[0.00–32.39]
9.52 A ± 1.13
[7.95–11.08]
9.01 A ± 1.39
[6.96–11.06]
59
59
70
59
59
n
26
26
28
23
23
Means with a common letter are not significantly different (p > 0.05). The variation in n observed among treatments
and recorded variables reflects the progressive decline in the number of surviving specimens over time, primarily
due to treatment effects. In addition, some individuals escaped from their individual containers and became
mixed, and others were lost because their diet became compromised by fungal growth. These specimens were
excluded from the sublethal dataset throughout the experiments.
Sublethal exposure to B. thuringiensis INTA Mo4-4 at LC30 and LC50 produced clear
effects on larval growth. Both concentrations significantly reduced mean larval weight and
body area compared with controls. Larval weight decreased from 0.37 ± 0.02 mg in controls
to 0.25 ± 0.02 mg in LC30 and 0.20 ± 0.02 mg in LC50 . Similarly, larval body area declined
from 1.36 ± 0.08 mm2 in controls to 0.95 ± 0.08 mm2 in LC30 and 0.82 ± 0.08 mm2 in LC50 .
In contrast, neither the duration of the larval stage nor that of the pupal stage differed
significantly among treatments. Pupation and adult emergence rates showed a decreasing
trend with increasing B. thuringiensis INTA Mo4-4 concentration (adult emergence: 42.79%
in controls; 26.29% in LC30 ; 11.78% in LC50 ), although these differences did not reach
statistical significance (p > 0.05) due to high individual variability. Likewise, no significant
differences were detected in pupal area, pupal weight, adult weight, or adult body area
across treatment groups.
3.3. Sex-Specific Effects
Pupal and adult measurements disaggregated by sex are presented in Table 3. While
statistical analysis (Bonferroni test) showed no significant differences in pupal or adult
weight and area among treatments within each sex, there was a mild increase in female
pupal weight and area at LC30 and LC50 compared to the control. Although non-significant
(p > 0.05), this trend suggests a potential differential physiological response between
genders under Bt stress.
Table 3. Sublethal effects on pupal and adult biological parameters with gender interaction.
Stage
Variable
Gender
Female
Weight
Male
Pupae
Female
Area
Male
Control (Mean ±
S.E. [min–max])
11.95 AB ± 0.56
[8.18–17.83]
9.30 A ± 0.60
[6.85–15.47]
10.13 BC ± 0.39
[7.20–14.80]
8.61 A ± 0.41
[5.90–12.00]
n
24
21
24
21
LC30 (Mean ± S.E.
[min–max])
12.77 B ± 0.62
[8.11–19.76]
9.47 A ± 0.67
[4.44–15.00]
10.73 BC ± 0.42
[7.90–13.50]
8.90 A ± 0.46
[5.70–13.70]
n
20
17
20
17
LC50 (Mean ± S.E.
[min–max])
12.35 AB ± 1.23
[9.27–16.10]
9.62 AB ± 0.97
[7.54–14.09]
11.34 C ± 0.85
[9.20–13.50]
9.20 AB ± 0.67
[6.60–12.70]
n
5
8
5
8
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Table 3. Cont.
Stage
Variable
Gender
Female
Weight
Male
Adult
Female
Area
Male
Control (Mean ±
S.E. [min–max])
n
10.45 AB ± 0.47
[6.97–14.18]
8.22 A ± 0.54
[7.76–12.91]
10.69 AB ± 0.39
[7.80–15.40]
8.97 A ± 0.46
[6.50–12.40]
24
18
24
18
LC30 (Mean ± S.E.
[min–max])
11.19 B ± 0.54
[6.87–16.71]
8.03 A ± 0.55
[3.47–12.34]
11.38 B ± 0.46
[7.70–16.00]
9.05 A ± 0.47
[4.90–11.00]
n
18
17
18
17
LC50 (Mean ± S.E.
[min–max])
10.96 AB ± 1.02
[8.34–13.82]
7.98 A ± 0.81
[5.67–12.21]
10.34 AB ± 0.86
[7.80–12.40]
9.06 AB ± 0.68
[6.90–12.70]
n
5
8
5
8
Means with a common letter are not significantly different (p > 0.05).
As shown in Table 3, the effects of sublethal concentrations during the pupal and
adult stages were influenced by gender. In pupae, female weight and body area tended
to increase slightly at LC30 and LC50 compared with the control, whereas male pupae
exhibited only minor, non-significant changes. Similarly, in adults, females showed a
slight increase in weight and body area under LC30 , while males displayed no significant variation across treatments. Statistical analysis using the Bonferroni test indicated
that most of these differences were not significant at the 0.05 level, highlighting subtle,
gender-specific responses.
3.4. Macromolecular Content
Biochemical analysis of surviving larvae (14 days post-exposure) is presented in Table 4.
Proteins and lipids were the most sensitive reserves, showing significant reductions when
expressed per individual in both LC30 and LC50 groups compared to the control (p < 0.05).
Conversely, the contents of soluble sugars and glycogen did not differ significantly across
treatments at the evaluated concentrations (Table 4).
Table 4. Macromolecule content of A. diaperinus larvae under control, LC30 and LC50 treatments.
Macromolecule
Control (Mean ± S.E.)
[min–max]
n
LC30
(Mean ± S.E.) [min–max]
n
LC50
(Mean ± S.E.)
[min–max]
n
Proteins
Lipids
Sugars
Glycogen
13.23 B ± 1.03 [10.43–17.13]
19.87 B ± 1.63 [14.04–27.56]
2.14 A ± 0.73 [0.89–3.38]
0.13 A ± 0.03 [0.07–0.20]
23
48
24
69
6.36 A ± 1.19 [5.46–7.75]
11.04 A ± 2.30 [8.37–13.31]
1.90 A ± 0.73 [1.66–2.14]
0.12 A ± 0.03 [0.10–0.13]
20
30
24
49
3.76 A ± 1.03 [2.67–6.00]
9.36 A ± 1.99 [6.22–13.64]
1.66 A ± 0.73 [1.63–1.68]
0.07 A ± 0.03 [0.03–0.13]
35
40
24
44
Means with a common letter are not significantly different (p > 0.05).
Values in µg/larva; mean ± standard error; n = number of larvae per pool; means
with same letter not significantly different, p > 0.05. The sample size (n) is variable as the
colorimetric reactions were performed on surviving residual specimens from each bioassay
date. Glycogen determinations include a higher number of replicates than glucose due to
the exclusion of some glucose measurements that did not meet quality control criteria.
The data reveal that proteins and lipids are the most sensitive macromolecular targets
of initial sublethal exposure. The significant decrease in these reserves is consistent with a
high energetic cost associated with the immune response or the repair of intestinal damage
caused by B. thuringiensis INTA Mo4-4. As shown in Table 4, the depletion of these energydense molecules was concentration-dependent, highlighting the metabolic stress imposed
by the entomopathogenic bacteria during the first 14 days of exposure.
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3.5. Long-Term Survival and Lethal Time (LT) Analysis
The effects of B. thuringiensis INTA Mo4-4 treatment on the survival probability of
A. diaperinus larvae over time are presented in Figure 1. Day 0 represents the end of the
initial bioassay under moist diet conditions and the transition to individual dry-diet tubes.
Individual survival records by insect, bioassay date, and treatment are available in the
Supplementary Material (Table S5: Global survival analysis).
Figure 1. Kaplan-Meier survival curves indicating survival probability as a function of time. The three
treatments under study were: CONTROL, LC30 and LC50 . The shaded areas represent the 95% confidence intervals for each survival curve. Cross marks on the curves indicate censored observations.
To maintain the rigor of the Kaplan-Meier analysis, individual specimens that could
not be monitored until natural death—due to fungal contamination (non-Bt related) or
technical incidents (e.g., escapes)—were treated as censored observations, as indicated by
cross marks in Figure 1.
A statistically significant difference in survival was found among the treatment groups
(χ2 = 109, df = 2, p < 2 × 10−16 ). The high χ2 value suggests a strong dose-dependent
impact of the Argentine Bt strain on the longevity of the population. Significant differences
between all pairs (Control vs. LC30 , Control vs. LC50 , and LC30 vs. LC50 ) were confirmed
through pairwise comparisons adjusted with the Bonferroni method (p < 0.05).
Survival percentiles, including the median survival (LT50 ) and the time to 90% mortality (LT90 ), are summarized in Table 5. The marked effect of the INTA Mo4-4 strain is evident
as the LT50 decreased dramatically from 116.58 days in the control to 14.28 and 4.19 days
for LC30 and LC50 , respectively. This indicates that even concentrations designed to be
sublethal in the short term trigger chronic lethal effects with an accelerated mortality rate
shortly after ingestion.
Furthermore, the chronic nature of these effects is reflected in the LT90 values. Although a small fraction of the population exhibited high resilience and a prolonged lifespan,
the time required to reach 90% mortality was reduced by approximately 24% and 40% in
the LC30 and LC50 groups, respectively, compared to the control. This confirms that the
physiological impairment sustained during the larval stage results in persistent biological
costs that significantly shorten the maximum life expectancy of the species.
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Table 5. Survival percentiles for each treatment group (days).
Treatment Group
LT50 (CI)
LT90 (CI)
Control
LC30
LC50
116.58 (106.95–121.35)
14.28 (10.78–18.92)
4.19 (3.43–6.00)
218.70 (164.63–427.88)
166.80 (149.85–331.07)
130.70 (112.85–152.54)
LT50 = median lethal time; LT90 = time at which 90% of individuals have died; CI = 95% confidence interval.
4. Discussion
This study investigated the initial sublethal effects of the Argentine Bacillus thuringiensis
strain INTA Mo4-4 on the development, fitness, and nutritional physiology of Alphitobius
diaperinus larvae. The interpretation of the observed effects is based on bioassay evidence
rather than on direct molecular or proteomic identification of individual Cry, Cyt, Vip, or
Vpa/Vpb proteins produced by INTA Mo4-4. Accordingly, the precise virulence factors
involved were not identified in the present study, and references to Cry toxins should be
understood within this experimental context. The results demonstrate profound and persistent chronic effects, particularly impacting larval growth, survival, and energy reserves.
These findings highlight the potential of initial sublethal B. thuringiensis concentrations to
disrupt the life cycle and fitness of this major poultry pest, offering insights for integrated
pest management (IPM) strategies.
Although the present bioassays were conducted under controlled laboratory conditions, with larvae maintained individually to prevent cannibalism, these constraints do not
preclude the ecological relevance of the observed effects. Under field conditions, where
food limitation and high larval densities may occur, exposure to B. thuringiensis could be
extended through indirect pathways such as cannibalism, a transmission route previously
demonstrated in tenebrionid beetles [25]. Such processes may contribute to sustained
sublethal exposure and reinforce the persistence of chronic effects in natural populations.
Consistent with this complexity, A. diaperinus exhibited pronounced intraspecific variability both within and among cohorts, reflecting the well-known resilience of tenebrionid
beetles. Cohorts, defined here as individuals hatching within a 24–48 h oviposition window, showed marked developmental asynchrony despite standardized rearing conditions.
Notably, within the same cohort and treatment, the interval between the first and last individuals reaching pupation extended up to 86 days (72 to 158 days), and younger cohorts
occasionally pupated earlier than older ones. Across bioassays, this variability was expressed as contrasting developmental outcomes (additional information is provided in the
Supplementary Materials) ranging from cases in which only control individuals completed
development to adulthood, to others in which both control and LC30 larvae reached the
adult stage, and, in some instances, survivors from all three treatments emerged as adults.
When survival occurred across treatments, two distinct patterns were observed: either
larval development converged toward control-like durations (LC50 ≈ LC30 ≈ control), or
treated larvae required longer developmental times than controls (LC50 > LC30 > control).
This high developmental plasticity, together with physiological traits characteristic of
beetle larvae—such as an acidic midgut environment that may limit crystal solubilization
and reduce Cry toxin activation [26,27]—likely contributes to the wide variability observed
in survival and developmental endpoints. Importantly, even in cases where apparent
developmental recovery was observed, sublethal effects persisted, as evidenced by delayed
mortality patterns reflected in LT50 and LT90 estimates, underscoring the chronic nature of
the effects detected. The reductions in larval weight, body area, and total protein and lipid
content observed after 14 days of exposure may result from reduced nutrient intake and/or
impaired nutrient absorption. Given the drastic reduction in LT50 observed in treated
larvae, the LC30 and LC50 used here are best interpreted as chronic lethal concentrations.
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The depletion of protein and lipid reserves serves as a biochemical proxy for the energetic
costs of surviving initial intoxication. While we recognize the absence of food consumption
measurements or gut histology as a limitation, the significant reduction in these energydense macromolecules suggests a metabolic trade-off, where energy is diverted from
growth toward detoxification or repair of intestinal damage. Nevertheless, although food
consumption and frass production were not directly quantified in this study, we cannot
rule out the possibility that larvae may have reduced their feeding activity. Similar patterns
have been documented in other species. Sutherland et al., 2003 [28] reported that starvation
in Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) larvae decreased midgut cell size
without causing lysis, and that individuals fed a Cry1Ac diet showed a feeding recovery
but ultimately reached a mean weight comparable to starved larvae.
Likewise, Luong et al., 2018 [29] suggested that behavioral avoidance of the toxin
contributed to the survival of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on
Bt-expressing plants, and Berdegué et al., 1996 [30] demonstrated significant avoidance
of Bt-treated diet by neonatal and third-instar Spodoptera exigua (Hübner) (Lepidoptera:
Noctuidae), with larvae consuming substantially more control diet across CryIC treatments.
Together, these findings support the possibility that reduced feeding—whether due to
behavioral avoidance or physiological stress—may contribute to the nutritional depletion
observed in our study.
Although the specific molecular identity of the pesticidal proteins in INTA Mo4-4 is
currently being elucidated via genomic sequencing, the localization of toxicity within the
spore-crystal pellet aligns with the typical pathology of B. thuringiensis in coleopterans.
The chronic effects observed here—reduced body mass and delayed mortality—suggest a
disruption of midgut integrity. In Tenebrionidae, this usually involves the binding of Cry
toxins to specific epithelial receptors, leading to septicemia or functional starvation [7,12,13].
In coleopteran insects, the mode of action of B. thuringiensis Cry toxins has been
closely associated with specific midgut receptors, particularly cadherins. In A. diaperinus,
Hua et al., 2014 identified the cadherin AdCad1 as a specific receptor for the Cry3Bb
toxin in larval midgut cells [12]. Subsequently, Park et al., 2014 demonstrated that a
fragment of the coleopteran cadherin DvCad1-CR8–10 synergistically enhances the toxicity
of Cry3Aa, Cry3Bb, and Cry8Ca, highlighting the functional role of cadherin-mediated
toxin binding in this species [7]. Although the present study did not directly investigate
Bt–receptor interactions or cadherin involvement, these previously described mechanisms
may contribute to, or be associated with, the biological effects observed here. In line with
this mechanistic framework, the patterns observed in our study are also compatible with
the well-documented disruption of the midgut epithelium caused by Bacillus thuringiensis
Cry toxins, which can compromise digestive efficiency and lead to nutritional depletion. A
substantial body of evidence supports this mechanism: Heckel 2020 [31] highlights pore
formation in epithelial membranes as the primary cause of Cry-induced cytotoxicity, with
ABC transporters and cadherins acting as key receptors whose disruption severely alters
gut integrity. Consistently, Bowling et al., 2017 [32] reported clear signs of intoxication in
Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) larvae exposed to various
insecticidal proteins, including swelling and sloughing of enterocytes and constriction of
midgut circular muscles. More recently, Ayra-Pardo et al., 2025 [33] showed that Cry1Ia-fed
Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) larvae exhibited extensive
midgut cell damage, impairment of digestion and nutrient absorption, and loss of the
peritrophic membrane. Together, these studies provide a coherent physiological explanation
for the nutritional depletion recorded in our bioassays.
Within the framework of the standardized bioassay, LC30 and LC50 correspond to
concentrations at which a substantial proportion of individuals survive the initial exposure
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period, while delayed mortality is revealed only through extended monitoring. Under
these conditions, approximately 70% and 50% of larvae survived the 14-day exposure to
LC30 and LC50 , respectively, and only surviving individuals were subsequently followed.
The limited acute mortality observed during exposure likely reflects individual variability
in susceptibility and behavioral responses to B. thuringiensis, including transient reductions
in feeding that may temporarily limit toxin ingestion. However, the pronounced, dosedependent reductions in LT50 values and the delayed mortality observed after transfer to a
toxin-free diet indicate that initial sublethal exposure induces chronic physiological damage
that is not fully expressed as acute mortality within standard short-term bioassays. Thus,
these concentrations, while permitting survival during the initial 14-day assay, should
be interpreted as chronic lethal doses due to the irreversible energetic and physiological
damage sustained by the larvae.
4.1. Impact on Larval Growth and Metabolism
The most significant sublethal effects observed were the severe reduction in larval
weight and body area (Table 2) and the dramatic depletion of key macromolecular reserves
(Table 4). The reduction in larval mass, which was proportional to the B. thuringiensis
concentration, is a classic sign of intoxication by Cry proteins. Upon ingestion, Cry toxins cause pore formation in the midgut epithelial cells, disrupting osmotic balance and
nutrient absorption [34,35]. The larvae likely compensated for this midgut damage and
nutrient malabsorption by diverting limited energy resources away from somatic growth
towards tissue repair, detoxification, and stress management, resulting in smaller body
size. The analysis of macromolecular content reinforces this interpretation. Protein content experienced the most severe depletion, followed by lipids. Proteins are crucial for
cellular maintenance, enzyme synthesis, and growth [36,37]. Their loss suggests severe
tissue damage and/or a failure in synthesizing new proteins, possibly due to reduced
energy input or direct Cry action on the gut [38]. The significant loss of lipids, one of
the primary long-term energy reserves and an important contributor to insect immune
function, indicates that larvae metabolized their reserves to fuel basic survival functions
and repair gut damage [28,39]. Importantly, the observed reductions in protein and lipid
content are consistent with metabolic stress and/or reduced nutrient intake but are not
presented here as direct evidence of immune activation or gut repair processes.
In A. diaperinus, total sugars and glycogen are rarely quantified, likely because they
represent minor components relative to total proteins and lipids. Based on findings in other
beetles where sugars are depleted during food deprivation [40,41], we hypothesize that
larvae surviving 14 days of exposure may have experienced a combination of self-imposed
fasting and/or impaired nutrient absorption. In our bioassays, no statistically significant
differences were detected in total sugars or glycogen, likely due to high internal variability;
nevertheless, trends were consistent with this physiological framework. These biochemical
disruptions are highly correlated with the impaired larval growth [42] (Table 2).
4.2. Chronic Effects on Development and Fitness
Despite the severe physiological stress, the duration of the larval stage did not change
significantly (Table 2). This lack of developmental delay, despite being smaller and metabolically stressed, contrasts with studies in other insects [43,44] and suggests that the surviving
A. diaperinus may have accelerated their development as a stress response to quickly exit
the toxic environment. However, this rapid development came at a clear cost to overall
fitness, as evidenced by the sharp, though statistically non-significant, decrease in pupation
and adult emergence rates (Table 2).
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The most striking long-term impact was the catastrophic reduction in survival time
(Figure 1, Table 5). The LT50 for the LC50 group was reduced from over 116 days to just
4 days, indicating that the chronic physiological damage sustained was severe enough to
drastically shorten the life expectancy of the surviving population. This finding underscores
the concept that sublethal exposure can act as a delayed mortality factor, a highly effective
result in pest control where persistent suppression is key [45–47].
4.3. Gender-Specific Responses
Females of A. diaperinus tend to have larger body size than males, an advantage for tolerating desiccation [48]. The absence of significant differences in final pupal and adult body
size suggests that survivors achieved a final size similar to the control group, potentially
through compensatory growth. However, Table 3 hints at subtle gender-specific responses.
While not statistically significant, female pupal and adult sizes trended slightly higher in
the LC30 group. This phenomenon, where females exhibit a positive size trend under mild
stress, may be related to sex-specific resource allocation for egg production [49,50].
4.4. Deformations by B. thuringiensis
Several cases of deformities induced by B. thuringiensis have been reported in species
such as Drosophila melanogaster Meigen (Diptera: Drosophilidae) [51], Galleria mellonella
(Linnaeus) (Lepidoptera: Pyralidae) [52] and Anastrepha fraterculus (Wiedemann) (Diptera:
Tephritidae) [53]. To the best of our knowledge, no reports have documented deformities
in beetles associated with B. thuringiensis exposure.
After extensive bioassays with INTA Mo4-4, we found no macroscopic evidence of
teratogenic effects in A. diaperinus. Notably, developmental damage was occasionally
observed in control specimens during molting, a physiologically stressful phase. Although alterations at the cellular level may occur—as revealed by micro-CT in Aedes
aegypti (Linnaeus) (Diptera: Culicidae) [54]—our conclusions are limited to the absence of
macroscopic deformities.
4.5. Implications for Pest Management
The results from this study confirm that the B. thuringiensis INTA Mo4-4 strain has significant potential for A. diaperinus control through both acute toxicity and potent sublethal
effects. Exposure to LC30 and LC50 concentrations leads to: reduced larval growth affecting subsequent reproductive capacity; severe metabolic stress compromising long-term
survival; and delayed yet high mortality.
These sublethal effects are highly relevant to field conditions in poultry houses, where
uneven application may result in larvae consuming non-lethal doses. The chronic toxicity
observed suggests that B. thuringiensis applications do not need to achieve 100% mortality
to be highly effective; instead, they function as potent growth and fitness suppressors.
Incorporating INTA Mo4-4 into an IPM program could therefore offer sustained pest
suppression by reducing the next generation’s population size and overall lifespan.
5. Conclusions
Initial sublethal exposure to Bacillus thuringiensis INTA Mo4-4 induced profound and
persistent physiological stress in Alphitobius diaperinus, with consequences extending far
beyond the initial exposure period. Larvae that survived the 14-day sublethal bioassays
exhibited marked reductions in body size, weight, and key macromolecular reserves,
particularly proteins and lipids, indicating severe impairment of growth and metabolic
homeostasis. These alterations reflect a state of chronic toxicity that likely compromises
the ability of individuals to cope with subsequent environmental challenges and may
negatively affect future reproductive performance.
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These effects were supported by statistically significant differences detected at multiple
biological levels. Significant reductions in larval area and weight were observed when
comparing both sublethal treatments (LC30 and LC50 ) with controls. In terms of survival, the
overall survival analysis revealed a significant effect of treatment, and subsequent pairwise
comparisons showed significant differences between all treatment combinations (control vs.
LC30 , control vs. LC50 , and LC50 vs. LC30 ). In addition, the nutritional composition of A.
diaperinus larvae differed significantly between controls and both treatments after 14 days
of dietary exposure, specifically in the total individual content of proteins and lipids.
Importantly, the physiological damage sustained during sublethal exposure translated
into pronounced delayed mortality, as evidenced by the drastic reduction in LT50 and LT90
values even after larvae were transferred to an uncontaminated diet. This demonstrates
that initial sublethal doses of B. thuringiensis can act as a powerful delayed-lethal factor,
substantially shortening lifespan and reducing population persistence despite apparent
short-term survival or partial developmental recovery.
Collectively, our results show that B. thuringiensis INTA Mo4-4 exerts its insecticidal
activity against A. diaperinus not only through direct lethality but also through sustained
chronic sublethal effects that disrupt growth, metabolism, survival, and overall fitness
across the life cycle. Although the present study does not include molecular or proteomic
characterization of individual Cry, Cyt, Vip, or Vpa/Vpb proteins and therefore does
not aim to dissect the specific mechanisms of action of B. thuringiensis in coleopterans,
our interpretation—grounded in previous reports and consistent with the entomological
scope of this work—supports two non-exclusive hypotheses, namely larval self-imposed
starvation as a survival strategy and Bt-induced midgut damage, and leaves no doubt
that B. thuringiensis INTA Mo4-4 induces chronic toxicity and delayed mortality. From
an applied perspective, these characteristics make INTA Mo4-4 a strong candidate for
the biocontrol of beetle pests, either as a standalone tool or as part of an integrated pest
management program. The ability to suppress populations through chronic toxicity and
delayed mortality is particularly relevant under field conditions, where exposure to nonlethal doses is common. While the molecular identification of its pesticidal proteins is
currently underway, the consistent and long-term impact on A. diaperinus development and
survival reported here validates this strain as a solid biocontrol agent for the development
of sustainable insecticides.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/insects17020213/s1, Table S1: Individual larval weight; Table S2:
Individual larval area; Table S3: Individual larval and pupal stage duration; Table S4: Pupae and
adults area and weight; Table S5: Global survival analysis.
Author Contributions: Conceptualization, G.I.A. and D.H.S.; methodology, G.I.A., L.C. and D.H.S.;
software, G.I.A., L.C. and D.H.S.; validation, G.I.A., L.C. and D.H.S.; formal analysis, G.I.A., L.C. and
D.H.S.; investigation, G.I.A., L.C. and D.H.S.; resources, D.H.S.; data curation, G.I.A., L.C. and D.H.S.;
writing—original draft preparation G.I.A.; writing—review and editing, D.H.S.; visualization, G.I.A.,
L.C. and D.H.S.; supervision, D.H.S.; project administration, D.H.S.; funding acquisition, D.H.S. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by INTA 2023-PD-L06-I116.
Data Availability Statement: Data are contained within the article.
Acknowledgments: SENASA, and especially Laura Maly, is gratefully acknowledged for her constant
support and for granting the flexibility needed to pursue scientific training. Gabriela Artave is
warmly thanked for her continued support in covering work responsibilities, which enabled the
completion of experimental work and data analysis. IMYZA and its Director, Mariana Viscarret, are
acknowledged for institutional support and encouragement. Melisa Perez is acknowledged for laying
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the foundations of this research through her previous work. Marcelo Berretta is thanked for donating
the glycogen used to generate the standard curve in larvae. The laboratory members Leila Ortiz, José
Niz, Maximiliano Torres and Augusto Salas are gratefully acknowledged for their valuable assistance
and support throughout this work.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
The following abbreviations are used in this manuscript:
LC
LT
IPM
Lethal Concentration
Lethal Time
Integrated Pest Management
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Panzer, G.W.F. Faunae Insectorum Germanicae Initia Oder Deutschlands Insecten; In den Felseckerschen Buchhandlung: Nürnberg,
Germany, 1793; Volume 37, pp. 1–60.
Goodwin, M.A.; Waltman, W.D. Transmission of Eimeria, Viruses, and Bacteria to Chicks: Darkling Beetles (Alphitobius diaperinus)
as Vectors of Pathogens. J. Appl. Poult. Res. 1996, 5, 51–55. [CrossRef] [PubMed]
Tufan-Cetin, O.; Cetin, H.A. Review of Biological and Sustainable Management Approaches for Alphitobius diaperinus, a Major
Pest in Poultry Facilities. Vet. Sci. 2025, 12, 158. [CrossRef] [PubMed]
Sammarco, B.C.; Hinkle, N.C.; Crossley, M.S. Biology and Management of Lesser Mealworm Alphitobius diaperinus (Coleoptera:
Tenebrionidae) in Broiler Houses. J. Integr. Pest Manag. 2023, 14, 2. [CrossRef]
Crickmore, N.; Berry, C.; Panneerselvam, S.; Mishra, R.; Connor, T.R.; Bonning, B.C. A structure-based nomenclature for Bacillus
thuringiensis and other bacteria-derived pesticidal proteins. J. Invertebr. Pathol. 2021, 186, 107438. [CrossRef]
Krieg, A.; Huger, A.M.; Langenbruch, G.A.; Schnetter, W. Bacillus thuringiensis var. tenebrionis: Ein neuer, gegenüber Larven von
Coleopteren wirksamer Pathotyp. Z. Angew. Entomol. 1983, 96, 500–508. [CrossRef]
Park, Y.; Hua, G.; Taylor, M.D.; Adang, M.J. A coleopteran cadherin fragment synergizes toxicity of Bacillus thuringiensis toxins
Cry3Aa, Cry3Bb, and Cry8Ca against lesser mealworm. Alphitobius diaperinus (Coleoptera: Tenebrionidae). J. Invertebr. Pathol.
2014, 123, 1–5. [CrossRef]
Sallet, L. Seleção de Estirpes de Bacillus thuringiensis Para o Controle de Alphitobius diaperinus (Coleoptera: Tenebrionidae). Ph.D.
Thesis, Universidad de Brasilia, Brasília, Brazil, 2013.
Pérez, M.P.; Benintende, G.B.; Sauka, D.H. Toxicity assessment of Bacillus thuringiensis strains for the control of the lesser
mealworm beetle Alphitobius diaperinus (Coleoptera: Tenebrionidae). Rev. Argent. Microbiol. 2025, 57, 375–379. [CrossRef]
Koc, S.; Polat, B.; Cengiz, A.; Kahraman, S.; Tufan Cetin, O.; Cetin, H. Effectiveness of some microbial biopesticides based on
Bacillus against lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae) under laboratory conditions. Fresenius Environ.
Bull. 2022, 31, 1537–1540.
Elgizawy, K.K.; Ashry, N.M. Efficiency of Bacillus thuringiensis strains and their Cry proteins against the Red Flour Beetle, Tribolium
castaneum (Herbst.) (Coleoptera: Tenebrionidae). Egypt. J. Biol. Pest Control 2019, 29, 94. [CrossRef]
Hua, G.; Park, Y.; Adang, M.J. Cadherin AdCad1 in Alphitobius diaperinus larvae is a receptor of Cry3Bb toxin from Bacillus
thuringiensis. Insect Biochem. Mol. Biol. 2014, 45, 11–17. [CrossRef]
Hasan, M.M.; Parween, S.; Reza, A.M.S.; Easmin, N. Response of Alphitobius diaperinus Panzer to Bacillus thuringiensis Berliner var.
kurstaki. Entomon 2002, 27, 441–446.
Behle, R.W.; McGuire, M.R.; Shasha, B.S. Effects of Sunlight and Simulated Rain on Residual Activity of Bacillus thuringiensis
Formulations. J. Econ. Entomol. 1997, 90, 1560–1566. [CrossRef]
Pérez, M.P. Factores de Virulencia de Bacillus thuringiensis y su Utilización Para el Control de Coleópteros de Alto Impacto en el
Sector Agropecuario. Ph.D. Thesis, Universidad Nacional de Buenos Aires, Buenos Aires, Argentina, 2017.
Iriarte, J.; Caballero, P. Biología y Ecología de Bacillus thuringiensis. In Bioinsecticidas: Fundamentos y Aplicaciones de Bacillus
thuringiensis en el Control Integrado de Plagas; Caballero, P., Ferré, J., Del Río, J.L., Eds.; Phytoma España: Valencia, Spain, 2001;
pp. 15–44.
Finney, D.J. Probit Analysis, 3rd ed.; Cambridge University Press: Cambridge, UK, 1971.
Nouri-Ganbalani, G.; Borzoui, E.; Abdolmaleki, A.; Abedi, Z.; Kamita, S.G. Individual and Combined Effects of
Bacillus Thuringiensis and Azadirachtin on Plodia Interpunctella Hübner (Lepidoptera: Pyralidae). J. Insect Sci. 2016, 16,
95. [CrossRef]
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
15 of 16
Abedi, Z.; Saber, M.; Vojoudi, S.; Mahdavi, V.; Parsaeyan, E. Acute, sublethal, and combination effects of azadirachtin and
Bacillus thuringiensis on the cotton bollworm, Helicoverpa armigera. J. Insect Sci. 2014, 14, 30. [CrossRef]
Esquivel, J.F.; Crippen, T.L.; Ward, L.A. Improved Visualization of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)—
Part I: Morphological Features for Sex Determination of Multiple Stadia. Psyche A J. Entomol. 2012, 2012, 328478. [CrossRef]
Van Herk, W.G.; Vernon, R.S.; Tolman, J.H.; Ortiz Saavedra, H. Mortality of a wireworm, Agriotes obscurus (Coleoptera: Elateridae),
after topical application of various insecticides. J. Econ. Entomol. 2008, 101, 375–383. [CrossRef]
Brogdon, W.G. Mosquito protein microassay—I. Protein determinations from small portions of single-mosquito homogenates.
Comp. Biochem. Physiol. B 1984, 79, 457–459. [CrossRef] [PubMed]
Anschau, A.; Caruso, C.S.; Kuhn, R.C.; Franco, T.T. Validation of the sulfo-phospho-vanillin (SPV) method for the determination
of lipid content in oleaginous microorganisms. Braz. J. Chem. Eng. 2017, 34, 19–27. [CrossRef]
Yuval, B.; Kaspi, R.; Shloush, S.; Warburg, M.S. Nutritional reserves regulate male participation in Mediterranean fruit fly leks.
Ecol. Entomol. 1998, 23, 211–215. [CrossRef]
Milutinovi´c, B.; Höfling, C.; Futo, M.; Scharsack, J.P.; Kurtz, J. Infection of Tribolium castaneum with Bacillus thuringiensis:
Quantification of Bacterial Replication within Cadavers, Transmission via Cannibalism, and Inhibition of Spore Germination.
Appl. Environ. Microbiol. 2015, 81, 8135–8144. [CrossRef]
Dow, J.A.T. Insect midgut function. In Advances in Insect Physiology; Evans, P.D., Wigglesworth, V.B., Eds.; Academic Press:
London, UK, 1987; Volume 19, pp. 187–328. [CrossRef]
Oppert, B. Protease interactions with Bacillus thuringiensis insecticidal toxins. Arch. Insect Biochem. Physiol. 1999, 42, 1–12.
[CrossRef]
Sutherland, P.W.; Harris, M.O.; Markwick, N.P. Effects of Starvation and the Bacillus thuringiensis Endotoxin Cry1Ac on the
Midgut Cells, Feeding Behavior, and Growth of Lightbrown Apple Moth Larvae. Ann. Entomol. Soc. Am. 2003, 96, 250–264.
[CrossRef]
Luong, T.T.A.; Zalucki, M.P.; Perkins, L.E.; Downes, S.J. Feeding behaviour and survival of Bacillus thuringiensis-resistant
and Bacillus thuringiensis-susceptible larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) exposed to a diet with Bacillus
thuringiensis toxin. Austral Entomol. 2018, 57, 1–8. [CrossRef]
Berdegué, M.; Trumble, J.T.; Moar, W.J. Effect of CryIC toxin from Bacillus thuringiensis on larval feeding behavior of Spodoptera
exigua. Entomol. Exp. Appl. 1996, 80, 389–401. [CrossRef]
Heckel, D.G. How do toxins from Bacillus thuringiensis kill insects? An evolutionary perspective. Arch. Insect Biochem. Physiol.
2020, 104, e21673. [CrossRef] [PubMed]
Bowling, A.J.; Pence, H.E.; Li, H.; Tan, S.Y.; Evans, S.L.; Narva, K.E. Histopathological effects of Bt and TcdA insecticidal proteins
on the midgut epithelium of Western corn rootworm larvae (Diabrotica virgifera virgifera). Toxins 2017, 9, 156. [CrossRef]
Ayra-Pardo, C.; Ramare, V.; Couto, A.; Almeida, M.; Martins, R.; Sousa, J.A.; Santos, M.J. The Proteolytic Activation, Toxic Effects,
and Midgut Histopathology of the Bacillus thuringiensis Cry1Ia Protoxin in Rhynchophorus ferrugineus (Coleoptera: Curculionidae).
Toxins 2025, 17, 84. [CrossRef]
Palma, L.; Muñoz, D.; Berry, C.; Murillo, J.; Caballero, P. Bacillus thuringiensis toxins: An overview of their biocidal activity. Toxins
2014, 6, 3296–3325. [CrossRef]
Melo, A.L.A.; Soccol, V.T.; Soccol, C.R. Bacillus thuringiensis: Mechanism of action, resistance, and new applications: A review.
Crit. Rev. Biotechnol. 2014, 36, 317–326. [CrossRef]
Corsetti, G.; Pasini, E.; Scarabelli, T.M.; Romano, C.; Singh, A.; Scarabelli, C.C.; Dioguardi, F.S. Importance of Energy, Dietary
Protein Sources, and Amino Acid Composition in the Regulation of Metabolism: An Indissoluble Dynamic Combination for Life.
Nutrients 2024, 16, 2417. [CrossRef]
Kureˇcka, M.; Kulma, M.; Petˇríˇcková, D.; Plachý, V.; Kouˇrimská, L. Larvae and pupae of Alphitobius diaperinus as promising protein
alternatives. Eur. Food Res. Technol. 2021, 247, 2527–2532. [CrossRef]
Khatami, L.; Ghassemi-Kahrizeh, A.; Hosseinzadeh, A.; Aramideh, S. The effect of sublethal doses of Bacillus thuringiensis Berliner
on Tuta absoluta (Meyrick) on resistant and susceptible tomato cultivars. Zemdirbyste-Agriculture 2023, 110, 367–374. [CrossRef]
Arrese, E.L.; Soulages, J.L. Insect fat body: Energy, metabolism, and regulation. Annu. Rev. Entomol. 2010, 55, 207–225. [CrossRef]
Laparie, M.; Larvor, V.; Frenot, Y.; Renault, D. Starvation resistance and effects of diet on energy reserves in a predatory ground
beetle (Merizodus soledadinus; Carabidae) invading the Kerguelen Islands. Comp. Biochem. Physiol. A 2012, 161, 122–129. [CrossRef]
Renault, D.; Hervant, F.; Vernon, P. Comparative study of the metabolic responses during food shortage and subsequent recovery
at different temperatures in the adult lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae). Physiol. Entomol. 2002,
27, 291–301. [CrossRef]
Chauhan, V.K.; Dhania, N.K.; Chaitanya, R.K.; Senthilkumaran, B.; Dutta-Gupta, A. Larval Mid-Gut Responses to Sub-Lethal
Dose of Cry Toxin in Lepidopteran Pest Achaea janata. Front. Physiol. 2017, 8, 662. [CrossRef]
Ghassemi-Kahrizeh, A.; Aramideh, S. Sub-lethal effects of Bacillus thuringiensis Berliner on larvae of Colorado potato beetle,
Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Arch. Phytopathol. Plant Prot. 2015, 48, 259–267. [CrossRef]
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
16 of 16
Eizaguirre, M.; Tort, S.; López, C.; Albajes, R. Effects of sublethal concentrations of Bacillus thuringiensis on larval development of
Sesamia nonagrioides. J. Econ. Entomol. 2005, 98, 464–470. [CrossRef] [PubMed]
Stenekamp, D.; Pringle, K.; Addison, M. Effect of genetically modified Bt maize in an artificial diet on the survival of Cydia
pomonella (Lepidoptera: Tortricidae). Fla. Entomol. 2016, 99, 200–205. [CrossRef]
Wang, L.Y.; Jaal, Z. Sublethal effects of Bacillus thuringiensis H-14 on the survival rate, longevity, fecundity and F1 generation
developmental period of Aedes aegypti. Dengue Bull. 2005, 29, 192–196.
Fast, P.G.; Régnière, J. Effect of exposure time to Bacillus thuringiensis on mortality and recovery of the spruce budworm
(Lepidoptera: Tortricidae). Can. Entomol. 1984, 116, 123–130. [CrossRef]
Renault, D.; Coray, Y. Water loss of male and female Alphitobius diaperinus (Coleoptera: Tenebrionidae) maintained under dry
conditions. Eur. J. Entomol. 2004, 101, 491–494. [CrossRef]
Celi, M.; Russo, D.; Vazzana, M.; Arizza, V.; Manachini, B. Does Bacillus thuringiensis Affect the Stress and Immune Responses of
Rhynchophorus ferrugineus Larvae, Females, and Males in the Same Way? Insects 2022, 13, 437. [CrossRef]
Belousova, I.; Pavlushin, S.; Subbotina, A.; Rudneva, N.; Martemyanov, V. Sex specificity in innate immunity of insect larvae.
J. Insect Sci. 2021, 21, 15. [CrossRef]
Nawrot-Esposito, M.P.; Babin, A.; Pasco, M.; Poirié, M.; Gatti, J.-L.; Gallet, A. Bacillus thuringiensis Bioinsecticides Induce
Developmental Defects in Non-Target Drosophila melanogaster Larvae. Insects 2020, 11, 697. [CrossRef] [PubMed]
Al-Mashhadani, M.M.A.; Al-Joboory, R.K.I. Effect of Bacillus thuringiensis on the biological aspects of the great waxworm Galleria
mellonella. Int. J. Health Sci. 2022, 6, 14886–14893. [CrossRef]
Martins, L.N.; de Souza Stori de Lara, A.P.; Ferreira, M.S.; Nunes, A.M.; Bernardi, D.; Leivas Leite, F.P.; Garcia, F.R.M. Biological
Activity of Bacillus thuringiensis (Bacillales: Bacillaceae) in Anastrepha fraterculus (Diptera: Tephritidae). J. Econ. Entomol. 2018, 111,
1486–1489. [CrossRef]
Alba-Tercedor, J.; Vilchez, S. Anatomical damage caused by Bacillus thuringiensis variety israelensis in yellow fever mosquito
Aedes aegypti (L.) larvae revealed by micro-computed tomography. Sci. Rep. 2023, 13, 8759. [CrossRef]
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cff085bcf9f99bb6e72c28460755f4b8
PDF Text
Text
Supplementary Information
Table S1: Individual larval weight
Bioassay date
Treatment
Mean
Standard
individual
larval
deviation
larval
weight
Average
Coefficient
of
variation
weight + (n)
December 5–19, 2023
CONTROL
0.41 (48)
0.28 (48)
0.37 (48)
0.42 (48)
0.37
0.25
January 17-31, 2024
0.28 (48)
0.23 (36)
0.24 (48)
January 27–February 10, 2024
0.24 (48)
December 5–19, 2023
0.22 (48)
0.18 (48)
0.17 (34)
January 1–15, 2024
January 17-31, 2024
January 27–February 10, 2024
LC30
December 5–19, 2023
January 1–15, 2024
LC50
January 1–15, 2024
January 17-31, 2024
0.06
16.80
0.02
0.20
8.24
0.02
11.90
0.21 (48)
January 27–February 10, 2024
Table S2: Individual larval area
Bioassay date + n
per treatment
Individual larval
area CONTROL
December 5–19, 2023
0.023
0.013
0.014
0.007
0.014
0.007
0.017
0.010
0.007
0.010
0.006
0.007
0.011
0.018
0.008
0.010
0.010
0.008
0.010
0.012
0.010
0.012
0.011
0.007
0.014
0.015
0.008
0.006
0.008
0.008
0.010
0.023
0.008
0.009
0.008
0.007
0.008
0.008
0.012
0.018
0.010
0.009
0.010
0.011
0.007
0.013
0.007
0.008
0.013
0.014
0.013
0.011
0.008
0.010
0.006
0.016
0.007
0.012
0.011
0.007
0.010
0.009
0.008
0.009
0.011
0.005
0.020
0.020
0.012
0.009
0.012
0.008
n CONTROL = 48
n LC30 = 48
n LC50 = 48
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Individual larval area
Individual larval
LC30
area LC50
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January 1–15, 2024
n CONTROL = 48
n LC30 = 36
n LC50 = 48
January 17-31, 2024
n CONTROL = 48
n LC30 = 48
n LC50 = 34
0.007
0.022
0.009
0.014
0.014
0.011
0.008
0.011
0.011
0.009
0.012
0.017
0.013
0.016
0.013
0.015
0.008
0.005
0.010
0.018
0.009
0.008
0.007
0.008
0.012
0.022
0.011
0.008
0.008
0.009
0.014
0.016
0.008
0.009
0.006
0.008
0.018
0.014
0.010
0.007
0.012
0.007
0.009
0.018
0.008
0.007
0.009
0.010
0.013
0.013
0.008
0.006
0.011
0.006
0.013
0.009
0.011
0.009
0.009
0.010
0.012
0.014
0.013
0.005
0.017
0.013
0.017
0.014
0.009
0.006
0.013
0.006
0.017
0.009
0.009
0.008
0.010
0.007
0.011
0.011
0.013
0.011
0.011
0.006
0.009
0.006
0.013
0.010
0.008
0.005
0.008
0.010
0.004
0.006
0.010
0.005
0.007
0.008
0.010
0.011
0.014
0.006
0.018
0.007
0.009
0.011
0.009
0.006
0.014
0.012
0.010
0.010
0.006
0.005
0.023
0.007
0.007
0.006
0.008
0.006
0.014
0.008
0.011
0.006
0.011
0.006
0.018
0.009
0.006
0.005
0.013
0.005
0.008
0.005
0.016
0.011
0.010
0.006
0.012
0.010
0.006
0.006
0.009
0.005
0.013
0.013
0.006
0.010
0.008
0.007
0.010
0.008
0.015
0.006
0.009
0.003
0.007
0.011
0.011
0.010
0.004
0.005
0.009
0.010
0.008
0.008
0.009
0.005
0.012
0.013
0.012
0.007
0.011
0.005
0.013
0.009
0.007
0.007
0.008
0.007
0.007
0.008
0.008
0.006
0.017
0.008
0.005
0.005
0.008
0.012
0.011
0.004
0.010
0.010
0.006
0.006
0.011
0.014
0.006
0.003
0.010
0.012
0.008
0.004
0.015
0.011
0.007
0.007
0.009
0.005
0.010
0.014
0.01
0.008
0.006
0.004
0.016
0.016
0.011
0.011
0.005
0.006
0.018
0.009
0.011
0.014
0.006
0.012
0.012
0.011
0.003
0.013
0.006
0.006
0.007
0.008
0.006
0.005
0.007
0.006
0.012
0.013
0.006
0.004
0.007
0.005
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�Insects 2026, 17, 213
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0.005
0.005
0.018
0.011
0.016
0.009
0.012
0.016
0.009
0.01
0.009
0.006
0.018
0.018
0.01
0.005
0.009
0.004
0.012
0.015
0.007
0.006
0.007
0.009
0.007
0.017
0.006
0.005
0.006
0.011
0.011
0.013
0.007
0.013
0.005
0.004
0.012
0.008
0.004
0.013
0.008
0.007
0.011
0.012
0.011
0.007
0.007
0.005
0.009
0.012
0.008
0.009
0.007
0.008
0.010
0.008
0.009
0.01
0.009
0.004
0.009
0.010
0.009
0.011
0.017
0.008
0.007
0.009
0.025
0.011
0.013
0.008
0.009
0.013
0.014
0.007
0.013
0.015
0.008
0.01
0.011
0.006
0.01
0.011
0.006
0.005
0.004
0.003
0.023
0.013
0.013
0.013
0.008
0.005
0.012
0.011
0.012
0.005
0.010
0.007
0.013
0.019
0.008
0.012
0.006
0.008
n LC30 = 48
0.009
0.020
0.013
0.012
0.006
0.014
n LC50 = 48
0.009
0.017
0.009
0.009
0.004
0.010
0.014
0.012
0.011
0.006
0.006
0.011
0.014
0.014
0.012
0.012
0.008
0.007
0.007
0.013
0.017
0.006
0.012
0.006
0.017
0.016
0.009
0.011
0.010
0.009
0.015
0.011
0.019
0.010
0.008
0.007
0.017
0.011
0.012
0.013
0.018
0.005
0.013
0.011
0.011
0.015
0.007
0.011
0.019
0.022
0.014
0.008
0.006
0.010
0.012
0.022
0.006
0.014
0.010
0.013
0.010
0.014
0.004
0.013
0.012
0.007
0.018
0.013
0.010
0.014
0.011
0.009
0.010
0.012
0.011
0.010
0.013
0.007
0.021
0.013
0.005
0.007
0.012
0.014
0.013
0.015
0.012
0.009
0.013
0.010
0.014
0.016
0.009
0.008
0.010
0.007
0.013
0.015
0.014
0.009
0.013
0.012
0.009
0.012
0.015
0.008
0.011
0.011
0.022
0.014
0.007
0.010
0.004
0.008
0.017
0.025
0.011
0.010
0.019
0.009
January 27–February
10, 2024
n CONTROL = 48
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
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Table S3: Individual larval and pupal stage duration
Treatment
Bioassay date
Days to pupation
Days to pupation
Pupal stage dura-
from sublethal
from egg hatching
tion
treatments
CONTROL
December 5–19,
71
96
89
114
5
7
2023
n=2-2-2
January 1–15,
87
105
5
2024
n=1-1-1
January 17-31,
61
61
79
79
5
7
2024
54
75
72
93
7
3
n=39-39-39
78
54
96
72
6
7
61
78
79
96
5
6
102
61
120
79
5
5
84
54
102
72
7
7
86
78
104
96
8
6
84
96
102
114
7
6
107
94
125
112
4
6
84
78
102
96
7
6
78
75
96
93
6
7
94
78
112
96
6
6
61
89
79
107
7
7
96
75
114
93
8
3
78
54
96
72
6
7
63
61
81
79
7
7
84
94
102
112
7
8
61
54
79
72
7
7
78
98
96
116
8
7
7
79
61
January 27–Feb-
51
51
69
69
2
7
ruary 10, 2024
74
88
92
106
7
nd
n=64-64-53
58
51
76
69
7
7
74
53
92
71
7
nd
51
44
69
62
5
2
74
56
92
74
7
nd
58
53
76
71
7
nd
51
44
69
62
nd
6
51
51
69
69
5
1
51
44
69
62
7
6
51
53
69
71
7
nd
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�Insects 2026, 17, 213
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LC30
December 5–19,
44
104
62
122
7
4
51
68
69
86
7
6
65
53
83
71
7
nd
68
68
86
86
8
4
51
68
69
86
1
6
51
68
69
86
7
6
53
113
71
131
nd
3
68
81
86
99
6
7
51
53
69
71
2
nd
51
51
69
69
7
1
58
76
76
94
7
8
53
51
71
69
nd
2
104
51
122
69
9
5
68
68
86
86
6
6
90
51
108
69
7
7
51
51
69
69
7
7
74
51
92
69
7
1
65
62
83
80
7
3
53
51
71
69
nd
2
56
51
74
69
7
7
62
51
80
69
6
1
NO
NO
NO
2023
n=0-0-0
January 1–15,
95
85
113
103
6
7
January 17-31,
98
61
116
79
9
5
2024
78
114
96
132
6
4
n=21-21-21
86
96
104
114
8
6
54
133
72
151
7
7
86
78
104
96
8
6
91
94
109
112
7
6
98
78
116
96
7
6
78
107
96
125
6
7
118
98
136
116
7
6
107
133
125
151
7
7
2024
n=2-2-2
7
158
140
January 27–Feb-
51
68
69
86
7
4
ruary 10, 2024
68
62
86
80
6
3
n=48-48-42
81
53
99
71
7
nd
53
58
71
76
nd
7
74
74
92
92
7
7
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
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LC50
December 5–19,
62
51
80
69
3
7
51
74
69
92
7
5
51
62
69
80
7
6
62
62
80
80
3
6
62
53
80
71
6
dead
51
79
69
97
7
7
81
53
99
71
7
nd
58
56
76
74
7
nd
58
58
76
76
7
7
56
58
74
76
nd
7
62
68
80
86
3
6
74
51
92
69
7
7
65
68
83
86
9
6
62
51
80
69
3
7
68
97
86
115
6
7
62
68
80
86
3
6
68
74
86
92
4
7
68
58
86
76
6
7
65
51
83
69
7
1
NO
NO
NO
NO
NO
NO
2023
n=0-0-0
January 1–15,
2024
n=0-0-0
January 17-31,
133
107
151
125
7
7
2024
78
98
96
116
8
9
n=5-5-5
107
125
7
January 27–Feb-
51
84
69
102
5
6
ruary 10, 2024
74
51
92
69
7
7
n=23-23-21
58
51
76
69
7
2
51
51
69
69
7
7
62
51
80
69
6
7
81
58
99
76
7
7
68
53
86
71
6
nd
68
53
86
71
6
5
68
53
86
71
6
nd
81
51
99
69
7
7
62
51
80
69
6
7
65
83
7
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�Insects 2026, 17, 213
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Table S4: Pupae and adults area and weight
Treatment
Bioassay
Gender
Pupal area
Pupal
Adult area
Adult
(mm2)
weight (mg)
(mm2)
weight (mg)
nd
nd
nd
8.70
8.42
January 17-
nd
11.10
13.69
11.90
12.70
31, 2024
M
5.90
6.97
6.50
6.36
n=24-34-34-
nd
9.60
8.79
9.60
8.08
33-33
M
7.40
7.04
7.30
6.22
M
10.20
11.80
9.30
9.77
F
9.70
11.72
11.40
10.16
M
8.70
9.57
9.30
8.56
M
9.30
9.55
9.90
8.81
M
8.40
8.37
8.90
7.59
M
8.20
9.53
8.40
8.50
F
13.50
15.20
13.20
12.68
nd
13.50
14.49
11.90
10.63
M
7.50
7.68
7.00
6.66
F
10.20
12.62
10.40
11.42
nd
9.20
8.86
10.70
10.80
F
10.40
11.72
9.50
8.61
nd
9.40
9.51
8.80
8.07
F
7.60
9.09
10.00
8.87
nd
9.90
9.81
12.90
13.10
F
11.20
14.63
8.30
7.99
F
7.20
8.76
10.80
8.97
nd
9.90
9.69
8.10
7.60
F
8.40
8.18
8.00
6.58
M
7.30
7.55
8.80
6.98
M
8.10
7.86
7.80
7.51
F
7.90
8.43
11.00
10.32
F
11.60
11.50
6.60
6.44
M
6.20
7.63
10.60
10.22
F
9.90
11.72
10.40
10.75
F
10.80
11.65
10.90
9.48
nd
10.40
10.13
8.30
6.08
M
7.70
7.00
11.40
10.25
nd
10.20
11.64
10.00
9.09
nd
10.40
10.50
nd
nd
date
CONTROL
January 1–
15, 2024
n=0-0-0-1-1
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
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January 27–
nd
9.20
9.63
9.90
8.74
February 10,
M
9.90
12.23
10.50
9.87
2024
nd
10.90
11.03
10.20
9.84
n=21-53-53-
F
10.40
12.21
10.50
10.83
46-46
nd
11.10
10.95
11.50
9.60
F
10.70
13.27
12.10
11.92
nd
10.50
12.14
10.80
10.99
nd
11.90
13.54
11.30
11.51
nd
10.60
11.74
11.90
10.31
nd
11.10
11.77
10.20
10.14
nd
14.80
19.39
15.10
16.75
nd
14.00
14.86
12.90
13.00
M
12.00
15.47
11.80
12.91
F
8.80
10.18
9.50
8.78
nd
12.20
11.31
12.10
11.71
nd
11.50
14.12
11.20
10.02
M
9.40
10.71
10.40
10.85
nd
10.00
11.63
10.60
10.19
nd
11.80
12.17
11.50
13.55
nd
14.40
16.55
12.10
11.77
nd
12.50
13.44
8.30
7.46
M
7.40
7.25
12.10
11.99
F
10.70
13.34
12.40
11.14
F
8.00
8.49
10.70
11.28
nd
12.70
14.15
8.20
6.51
M
12.00
13.10
12.90
15.00
M
10.90
12.56
9.20
6.97
nd
11.50
11.84
11.80
12.00
nd
10.70
11.52
13.70
12.51
M
8.10
7.16
10.50
11.00
nd
14.90
17.59
15.40
14.90
F
9.30
10.42
11.30
12.14
nd
12.20
13.92
13.10
14.18
nd
13.20
17.87
10.40
10.20
nd
7.70
7.89
11.80
10.83
nd
11.70
13.17
9.60
7.76
nd
10.80
10.23
10.40
10.54
M
7.00
6.85
11.70
9.75
F
11.10
12.76
10.60
10.12
nd
10.90
10.52
12.20
13.00
F
11.50
16.46
14.40
16.13
F
10.40
13.74
9.70
8.25
F
14.80
17.83
10.60
10.42
F
9.50
11.18
11.90
11.49
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
9 of 19
LC30
nd
13.70
14.67
10.20
9.36
M
9.20
9.43
9.90
9.51
nd
10.70
11.27
nd
nd
nd
10.30
12.03
nd
nd
F
9.60
11.59
nd
nd
nd
12.90
14.06
nd
nd
nd
15.30
17.53
nd
nd
nd
11.20
13.13
nd
nd
nd
10.90
10.55
nd
nd
January 1–
F
9.60
11.03
9.90
10.31
15, 2024
nd
9.80
11.07
8.60
9.59
January 17-
M
6.90
6.04
6.70
4.51
31, 2024
M
7.20
6.29
6.80
5.62
n=14-16-16-
M
8.20
8.87
8.60
7.10
15-15
M
9.50
9.73
9.20
8.32
F
9.20
10.35
9.50
9.40
nd
9.40
9.83
9.00
9.00
F
8.80
9.77
8.90
8.86
M
5.70
4.44
4.90
3.47
nd
8.60
7.65
8.50
7.01
F
9.50
9.03
9.30
8.53
M
8.30
9.79
10.40
10.37
F
8.90
11.21
10.90
8.90
M
8.90
9.69
7.70
7.32
F
7.90
8.11
8.00
6.87
F
8.60
8.25
9.70
9.44
F
9.40
10.94
nd
nd
January 27–
nd
nd
nd
12.60
11.62
February 10,
nd
11.20
13.83
15.10
14.45
2024
F
13.50
16.98
10.10
7.29
n=22-41-41-
M
8.50
8.87
10.70
12.34
42-42
nd
13.10
14.78
9.70
9.60
M
13.70
15.00
14.30
14.90
nd
14.70
16.73
12.80
13.70
nd
12.60
15.06
12.20
11.70
M
7.80
9.00
9.90
8.20
nd
12.30
13.55
13.00
11.60
F
13.30
19.76
16.00
16.71
nd
12.60
14.37
11.40
12.80
nd
9.00
9.96
9.00
8.75
nd
13.00
15.42
15.50
17.12
F
15.00
14.93
13.70
12.90
F
11.80
17.79
14.10
15.02
n=1-2-2-2-2
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
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LC50
M
9.30
9.64
10.60
11.32
M
9.70
10.43
8.40
8.42
M
12.00
13.81
11.40
12.19
F
9.30
11.11
10.50
8.94
M
8.70
10.17
11.00
11.02
nd
10.00
9.92
9.90
10.19
nd
9.70
10.77
10.00
8.94
F
8.80
10.21
8.80
8.82
nd
11.00
12.24
9.60
9.06
M
8.60
9.62
9.10
9.08
F
9.60
11.82
10.50
10.07
F
12.30
13.20
8.80
8.48
nd
12.70
14.61
10.90
10.66
F
10.20
12.73
12.90
12.45
nd
12.10
14.27
10.60
10.45
nd
8.00
7.62
12.70
15.10
nd
11.70
17.43
11.30
11.49
nd
11.50
13.25
11.80
14.85
F
13.30
17.00
12.80
12.62
nd
14.00
14.60
13.10
13.75
F
12.40
15.82
10.30
10.61
nd
11.30
11.93
7.70
5.98
M
7.70
6.90
10.30
10.37
M
10.60
12.72
13.40
13.33
F
13.20
15.28
10.90
10.34
nd
9.90
12.00
10.60
9.57
NO
NO
NO
NO
NO
January 17-
M
6.60
7.54
7.40
6.59
31, 2024
M
7.60
7.91
6.90
6.92
n=4-4-4-4-4
M
8.70
8.55
8.20
7.17
M
8.30
8.46
9.30
7.14
January 27–
nd
9.10
9.33
9.30
8.13
February 10,
M
8.20
8.50
8.80
7.52
2024
nd
11.10
14.37
12.30
12.43
n=9-22-22-
nd
12.60
14.44
13.50
12.52
19-19
F
9.70
9.63
7.80
9.00
F
9.20
9.27
8.90
8.34
F
12.40
13.68
11.30
12.41
M
12.70
12.29
10.30
10.65
M
9.00
9.65
8.90
5.67
F
11.90
13.05
11.30
11.25
M
12.50
14.09
12.70
12.21
January 1–
15, 2024
n=0-0-0
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�Insects 2026, 17, 213
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nd
12.60
14.35
7.40
6.50
nd
8.00
7.16
14.60
14.31
nd
14.80
17.22
11.10
11.95
nd
12.50
13.20
10.70
12.68
nd
12.20
14.78
9.50
9.83
nd
13.00
15.23
14.90
15.77
nd
10.20
11.12
14.90
15.22
nd
11.90
13.88
12.40
13.82
nd
14.60
18.46
nd
nd
nd
15.30
17.18
nd
nd
F
13.50
16.10
nd
nd
Table S5: Global survival analysis
Treatment
Bioassay
Survival (days)
Censorship code
date
CONTROL
December
33
14
1
1
5–19, 2023
19
26
1
1
n=48
140
5
1
1
21
21
1
1
21
1
1
1
23
1
1
1
19
28
1
1
1
1
1
1
40
28
1
1
40
168
1
1
19
1
1
1
28
35
1
1
30
1
1
1
37
28
1
1
19
35
1
1
35
16
1
1
26
14
1
1
19
14
1
1
33
1
1
1
37
37
1
1
19
19
1
1
28
19
1
1
21
16
1
1
14
9
1
1
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
12 of 19
January 1–
10
13
1
1
15, 2024
10
10
1
1
n=13
6
24
1
1
13
154
1
1
20
10
1
1
13
13
1
1
1
13
January 17-
165
122
1
1
31, 2024
165
165
1
1
n=48
122
122
1
1
165
8
1
1
1
97
1
0
122
165
1
1
122
165
1
1
125
165
1
1
6
165
1
1
122
138
1
1
165
165
1
1
122
165
1
1
1
165
1
1
1
165
1
1
122
165
1
1
122
165
1
1
165
1
1
1
122
8
1
1
122
174
1
1
125
174
1
1
122
1
1
1
125
174
1
1
122
380
1
1
125
477
1
1
January 27–
3
36
1
0
February
36
36
0
0
10, 2024
12
43
1
0
n=127
5
36
1
0
36
115
0
1
97
36
0
0
112
115
1
1
36
155
0
1
122
155
1
1
36
155
0
1
112
155
1
1
122
36
1
0
36
36
0
0
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�Insects 2026, 17, 213
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36
155
0
1
112
155
1
1
36
155
0
1
36
155
0
1
122
164
1
1
79
36
0
0
36
43
0
0
36
115
0
0
122
5
1
1
122
112
1
1
36
115
0
1
128
78
1
0
36
36
0
0
5
36
1
0
36
36
0
0
155
3
1
1
80
78
1
0
112
112
1
1
36
3
0
1
36
3
0
1
5
164
1
1
112
5
1
1
36
50
0
1
155
36
1
0
36
164
0
1
155
316
1
1
155
36
1
0
78
50
1
1
5
370
1
1
3
36
1
0
93
449
1
1
155
36
1
0
78
449
0
1
155
36
1
0
8
457
1
1
36
457
0
1
155
36
1
0
78
483
0
1
155
87
1
0
155
492
1
1
112
492
1
1
112
80
1
1
155
492
1
1
36
36
0
0
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
14 of 19
155
36
1
0
36
36
0
0
112
36
1
0
155
492
1
1
3
521
1
1
36
36
0
0
0
36
LC30
December
23
14
1
1
5–19, 2023
26
26
1
1
n=48
12
12
1
1
30
1
1
1
5
19
1
1
19
12
1
1
12
35
1
1
1
12
1
1
5
16
1
1
21
16
1
1
21
12
1
1
19
19
1
1
14
1
1
1
16
19
1
1
1
33
1
1
35
12
1
1
21
5
1
1
21
23
1
1
16
23
1
1
14
19
1
1
28
19
1
1
14
21
1
1
19
1
1
1
21
16
1
1
January 1–
1
3
1
1
15, 2024
1
3
1
1
n=37
1
10
1
1
1
6
1
1
1
6
1
1
1
3
1
1
1
148
1
1
1
13
1
1
1
6
1
1
1
13
1
1
1
8
1
1
1
154
1
1
3
13
1
1
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
15 of 19
6
3
1
1
15
3
1
1
10
7
1
1
3
7
1
1
6
8
1
1
1
3
January 17-
1
1
1
1
31, 2024
125
1
1
1
n=48
122
182
1
1
122
165
1
1
1
1
1
1
4
4
1
1
132
165
1
1
6
6
1
1
122
379
1
1
122
6
1
1
1
1
1
1
1
1
1
1
1
1
1
1
125
8
1
1
122
122
1
0
132
8
1
1
122
125
1
1
125
152
1
1
165
152
1
0
11
11
1
1
1
39
1
1
4
4
1
1
125
1
1
1
152
1
1
1
January 27–
2
12
1
1
February
4
154
1
1
10, 2024
115
115
0
1
n=96
4
18
1
1
112
4
1
1
4
4
1
1
2
4
1
1
112
4
1
1
122
115
1
1
4
115
1
1
112
4
1
1
122
154
1
1
122
36
1
0
36
2
0
1
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
16 of 19
LC50
36
164
0
1
122
164
1
1
2
36
1
0
36
164
0
1
4
4
1
1
122
384
1
1
4
56
1
1
112
384
1
1
122
384
1
1
122
36
1
0
2
420
1
1
122
4
1
1
122
457
1
1
128
4
1
1
2
115
1
1
28
483
1
1
154
500
1
1
36
77
0
0
112
507
1
1
112
2
1
1
154
507
1
1
2
2
1
1
112
82
1
0
4
4
1
1
154
4
1
1
28
4
1
1
112
2
1
1
4
511
1
1
36
511
0
1
154
4
1
1
2
2
1
1
4
4
1
1
154
521
1
1
2
4
1
1
December
1
12
1
1
5–19, 2023
1
5
1
1
n=48
14
1
1
1
33
21
1
1
1
12
1
1
23
14
1
1
1
26
1
1
1
5
1
1
14
12
1
1
1
33
1
1
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
17 of 19
12
21
1
1
21
9
1
1
23
23
1
1
12
12
1
1
1
26
1
1
26
28
1
1
19
5
1
1
1
1
1
1
5
5
1
1
1
1
1
1
19
26
1
1
12
23
1
1
19
1
1
1
5
28
1
1
January 1–
3
1
1
1
15, 2024
3
1
1
1
n=48
3
1
1
1
3
1
1
1
6
1
1
1
6
1
1
1
6
1
1
1
3
1
1
1
10
1
1
1
49
1
1
1
122
1
1
1
6
1
1
1
10
1
1
1
8
1
1
1
10
1
1
1
3
1
1
1
8
1
1
1
8
1
1
1
8
1
1
1
13
1
1
1
8
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
January 17-
1
1
1
1
31, 2024
1
4
1
1
n=34
1
1
1
1
4
4
1
1
152
1
1
1
125
1
1
1
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
18 of 19
132
1
1
1
4
22
1
1
28
138
1
1
11
4
1
1
1
1
1
1
1
138
1
1
6
6
1
1
6
4
1
1
4
4
1
1
4
4
1
1
39
1
1
1
January 27–
2
2
1
1
February
28
2
1
1
10, 2024
115
2
1
1
n=80
2
4
1
1
2
4
1
1
4
36
1
0
36
2
0
1
7
2
1
1
4
2
1
1
2
4
1
1
112
2
1
1
36
66
0
1
4
2
1
1
2
154
1
1
18
77
1
0
2
154
1
1
122
154
1
1
4
2
1
1
4
2
1
1
4
36
1
0
4
12
1
1
122
4
1
1
2
154
1
1
2
154
1
1
112
36
1
0
12
2
1
1
2
4
1
1
163
115
1
1
4
154
1
1
4
4
1
1
12
115
1
1
383
115
1
1
4
36
1
0
https://doi.org/10.3390/insects17020213
�Insects 2026, 17, 213
19 of 19
483
7
1
1
8
36
1
0
36
154
0
1
4
507
1
1
2
36
1
0
77
521
1
1
154
2
1
1
Abbreviations
nd = no data
F = female
M = male
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://doi.org/10.3390/insects17020213
�
Dublin Core
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Title
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Trabajos, Tesis, Tesinas
Dublin Core
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Title
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Initial Sublethal Exposure to an Argentine <em>Bacillus thuringiensis</em> Strain Induces Chronic Toxicity and Delayed Mortality in <em>Alphitobius diaperinus </em>(Coleoptera: Tenebrionidae)
Creator
An entity primarily responsible for making the resource
Antonuccio, Gisele
Sauka, Diego
Candás, Lucas
Source
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Publicado en <em>Insects</em> (2026) 17 (2) 213
Date
A point or period of time associated with an event in the lifecycle of the resource
2026
Language
A language of the resource
En
Type
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Artículo
Identifier
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B.S.0201 - B.S. supl.
Abstract
A summary of the resource.
En este trabajo, evaluamos los efectos subletales iniciales de una cepa argentina de Bacillus thuringiensis en larvas de Alphitobius diaperinus tras 14 días de exposición dietética y realizamos un seguimiento de los insectos a lo largo de su ciclo de vida para evaluar la toxicidad crónica.
Control de Plagas
-
https://biblioteca.senasa.gob.ar/files/original/0367ff0a80b02f6e867e5b989b3663e0.pdf
ac5b5747a90b2ae0418d8f8cb6d6334c
PDF Text
Text
Sublethal effects of an Argentine Bacillus thuringiensis strain on the development and fitness of
Alphitobius diaperinus (Coleoptera: Tenebrionidae)
Gisele Ivonne Antonuccio1,2* and Diego Herman Sauka1,3
1
Instituto Nacional de Tecnología Agropecuaria (INTA), Instituto de Microbiología y Zoología Agrícola (IMYZA), Hurlingham, Buenos Aires, Argentina
2 Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), Buenos Aires, Argentina
3 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
*Correspondence: antonuccio.gisele@inta.gob.ar
INTRODUCTION & AIM
RESULTS & DISCUSSION
• Alphitobius diaperinus (lesser mealworm) is a significant pest affecting broiler • Mortality results: LC30 (µg/ml)= 69 [62 – 84] CV=15%; LC50 (µg/ml) = 136 [118 –
and layer poultry facilities.
155] CV=12%; Slope: 1.82; χ² (4 df) = 4.57.
• Its life cycle (Fig. 1) occurs entirely within poultry litter and manure, leading to
structural damage, bird injuries, and serving as a reservoir for microbial • Table 1 presents the sublethal effects of different lethal concentrations (LC30 and
pathogens.
LC50) of B. thuringiensis INTA Mo4-4 on key developmental parameters of A.
• Current control strategies rely on the use of chemical insecticides and the
diaperinus larvae, pupae and adults.
periodic replacement of litter.
Table 1. Partial life table of A. diaperinus. The average, minimum, and maximum values are
• The INTA Mo4-4 strain of Bacillus thuringiensis, identified through a nationwide indicated for each parameter. Means with a common letter are not significantly different (p >
screening of native Argentine strains, has shown high lethality against A. 0.05).
diaperinus larvae.
Measured variable
CONTROL
LC30
LC50
Larval stage duration
from hatching (days) 94.69A [78.31 – 105.00] 101.13A [80.92 -114.48] 122.60A [96.00 – 151.00]
Larval stage duration
since the end of Bt
intake (days)
76.69A [60.31 – 87.00] 83.13A [62.92 - 96.48] 104.60A [78.00 – 133.00]
Pupal stage duration
(days)
5.71A [5.00 - 6.36]
6.35A [5.93 - 6.62]
6.95A [6.29 - 7.60]
Life cycle of Alphitobius
diaperinus
Fig. 1 . Life cycle of A. diaperinus. (a) Eggs in clusters; (b) early-stage larva with molted exoskeleton;
(c) advanced-stage larva; (d) larva preparing to pupate; (e) A. diaperinus pupa; (f, g) pupa undergoing
progressive molting into an adult; (h) newly emerged whitish adult; (i) sclerotizing adult with a
brown coloration; and (j) fully mature black adult.
The aim of this work was to evaluate the sublethal effects of B. thuringiensis INTA
Mo4-4 on the development, survival, and fitness of A. diaperinus to assess its
potential as an effective biocontrol agent in poultry facilities.
METHOD
In figure 2, a schematic presentation of the two-stage workflow used to quantify
both lethal and sublethal effects of the INTA Mo4-4 strain on A. diaperinus larvae is
shown.
Larval area (mm2)
1.32B [1.17 - 1.67]
0.97A [0.87 - 1.06]
0.82A [0.71 - 0.95]
Larval weight (mg)
0.37B [0.28 - 0.42]
0.25A [0.23 -0.28]
0.20A [0.17 - 0.22]
Pupal area (mm2)
9.32A [5.90 - 13.50]
8.44A [5.70 - 9.50]
7.80A [6.60 - 8.70]
Pupal weight (mg)
10.08A [6.97 - 14.63]
8.75A [4.44 - 11.21]
8.12A [7.54 - 8.55]
Adult area (mm2)
9.63A [6.50 - 13.20]
8.54A [4.90 - 10.90]
7.95A [6.90 - 9.30]
Adult weight (mg)
8.97A [6.08 - 13.10]
7.65A [3.47 - 10.37]
6.96A [6.59 - 7.17]
Larval effects:
• LC30 treatment: Reduced weight and area compared to control .
• LC50 treatment: Further reduced weight and area.
• Statistically significant differences compared to control.
Developmental periods:
• Larval and pupal stages were prolonged under higher treatment
concentrations.
• Trends observed, but differences not statistically significant.
Pupal and adult fitness:
• Pupal weights and sizes decreased as treatment concentrations increased.
• Adult weights and body areas followed a similar decreasing trend, with lower
values observed at higher concentrations.
• Trends observed, but differences not statistically significant.
CONCLUSION
• B. thuringiensis INTA Mo4-4 induces both mortality and sublethal effects on A.
diaperinus.
• Sublethal effects include reduced larval size and weight, with potential
implications for pest development and fitness.
• Findings highlight the potential of B. thuringiensis INTA Mo4-4 as a bioinsecticide
for controlling A. diaperinus, with insights for further development.
FUTURE WORK
Fig. 2. First series of bioassays to determine lethal concentrations 30 and 50; second series of bioassays
to study the sublethal effects themselves. Monitoring of larvae that subsequently pupated and
developed into adults. Weight and area were recorded at each stage of the life cycle.
• Once individual death date records are complete, we will estimate key
mortality metrics (LT50 and LT90) to evaluate the time-dependent effectiveness
of treatments.
• Biochemical analyses will be performed on surviving larvae to assess potential
differences in metabolic reserves (e.g., proteins, lipids, glucose, glycogen)
across treatments.
• Further bioassays will examine the combined effects of B. thuringiensis INTA
Mo4-4 and Enterobacter sp. INTA AN1-1, the most abundant bacteria isolated
from the culturable gut microbiota of A. diaperinus.
https://sciforum.net/event/IECE2025
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Trabajos, Tesis, Tesinas
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Sublethal effects of an Argentine <em>Bacillus thuringiensis</em> strain on the development and fitness of <em>Alphitobius diaperinus</em> (Coleoptera: Tenebrionidae)
Creator
An entity primarily responsible for making the resource
Antonuccio, Gisele
Sauka, Diego
Date
A point or period of time associated with an event in the lifecycle of the resource
2025
Language
A language of the resource
En
Type
The nature or genre of the resource
Póster académico
Identifier
An unambiguous reference to the resource within a given context
B.S.0202
Abstract
A summary of the resource.
Trabajo presentado en: The 2nd International Electronic Conference on Entomology. 19-21 May 2025. El objetivo de este trabajo fue evaluar los efectos subletales de B. thuringiensis INTA Mo4-4 sobre el desarrollo, la supervivencia y la aptitud de A. diaperinus para determinar su potencial como agente de biocontrol eficaz en instalaciones avícolas.
Aves
Control de Plagas