BUSCADOR RECOLECTA

Hyposoter didymator (Thunberg) (Hymenoptera: Ichneumonidae) and baculovirus (BV) might be used jointly to provide effective control of the Spodoptera genus. The literature has mostly covered the safe compatibility between natural enemies and BV-based insecticides, but research on the potential dispersal of BV by natural enemies is lacking. Thus, the goal of this manuscript was to ascertain if H. didymator was able to disperse the broad host range of Autographa californica nucleopolyhedrovirus (AcMNPV) to Spodoptera littoralis and Spodoptera exigua in choice and non-choice conditions and whether the preference of the parasitoid by one of these noctuids could mediate this dispersion. It was previously needed to improve the rearing of the parasitoid in the laboratory, concerning the optimal host age and length of parasitization, parasitoid competition, and influence of parasitization on the longevity of females. The best rearing conditions for S. littoralis are collective parasitization of mature L3 larvae for 24 h, after at least one day of copulation. Hyposoter didymator transmits AcMNPV to both lepidopterans, but its efficiency is mediated by host preference and the pathogenicity of the BV in each host. In this particular case, H. didymator as well as AcMNPV showed a clear preference towards S. exigua., This research was funded by MCIN/AEI/10.13039/501100011033, grants AGL2017-83498-C2-2-R, PID2020-117062RB-C22 and IJC2018-035042-I, and by Ministerio de Hacienda de Paraguay/Programa nacional de becas de postgrado en el exterior D. Carlos Antonio López, grant 01/2019.

Background: Alphabaculoviruses are Lepidoptera-specific virulent pathogens that infect numerous pests, including the Spodoptera complex. Due to their low environmental persistence, the traditional use of Alphabaculoviruses as bioinsecticides consist in high-rate spray applications with repeated treatments. Several abiotic and biotic factors can foster its dispersion,
promoting their persistence in the agroecosystem. Amongst biotic factors, predatory arthropods can disperse the viruses by
excretion after preying on infected individuals. Therefore, this study focused on promoting predator's ingestion of nucleopolyhedrovirus (NPV)-treated diets, and the later exposition of the insect host to leaf surfaces contaminated with predator
excreta. The virus–host–predator system studied was Spodoptera littoralis nucleopolyhedrovirus (SpliNPV), Spodoptera littoralis (Boisduval) and Nesidiocoris tenuis (Reuter). The infective potential of N. tenuis feces and the retention time of SpliNPV were
assessed under laboratory conditions after feeding on treated diets (sucrose solution and Ephestia kuehniella eggs).
Results: Mortality of S. littoralis larvae was lower via N. tenuis excretion than in positive control (spray application) in the first
infection cycle, together with a delay in host death. In the second infection cycle, both SpliNPV-treated diets triggered 100%
mortality. Both diets allowed the transmission of SpliNPV, with a faster excretion via sucrose solution compared to
E. kuehniella eggs. SpliNPV remained in N. tenuis digestive tract and was viable after excretion at least for 9 days for both diets.
Conclusions: This study demonstrated the potential of the predator N. tenuis as inoculative agent of baculoviruses, representing a new alternative that, along with inundative applications, might contribute to improve pest management strategies., This study was funded by the Spanish Ministry of Science and Innovation through the State Research Agency with the projects entitled: AGL2017-83498-C2-2-RB ‘Development of effective baculovirus-based insecticides with a broad spectrum of hosts and advances towards its integration in IPM programs’ and PID2020-117062RB-C22 ‘Exploiting entomophagous insects to enhance the control of Spodoptera spp through the use of baculovirus’. OG Gutiérrez-Cárdenas acknowledges the Government of Mexico and the National Council of Humanities, Science and Technology (CONAHCYT) for the PhD research grant number 717840-2018.

Spodoptera littoralis (Boisduval, 1833) and Spodoptera exigua (Hübner, 1808) are two noctuid pests whose marked polyphagy makes them especially harmful to agriculture in southeastern Spain, sometimes appearing individually and on other occasions in coexistence. Currently they are basically controlled by chemical control, giving rise to resistance phenomena, which is why various control strategies have been investigated and applied, including biological control.

Hyposoter didymator (Thunberg, 1822) and Chelonus inanitus (Linnaeus, 1767) are two koinobiont endoparasitoids, larval and ovolarval respectively, whose females oviposit in the host, where the larva develops and feeds on its tissues, exits the host before spinning a silk cocoon, and emerge as an adult. Both parasitoids can be used to control species of the genus Spodoptera, especially S. exigua. On the other hand, another type of biological control against noctuids is baculoviruses, entomopathogens that are marketed as microbial insecticides.

In this Thesis, we have worked with the broad-spectrum baculovirus AcMNPV-Autographa californica multiple nuclear polyhedrosis virus, and those specific to S. exigua (SeMNPV-Spodoptera exigua nuclear polyhedrosis virus) and S. littoralis (SpliMNPV-Spodoptera littoralis multiple nuclear polyhedrosis virus), with the main objective of studying, under laboratory conditions, their dispersal by the parasitoids H. didymator and C. inanitus, also analyzing their interactions.

First, the development of C. inanitus was studied, both in its natural host S. littoralis and in the alternative host Ephestia kuehniella (Zeller, 1879), with the aim of improving its breeding in the laboratory. It was observed that the longevity of C. inanitus adults was significantly shorter in the presence of eggs from each of the hosts compared to the absence of eggs. Regarding reproduction, it was shown that the reproduction of the parasitoid C. inanitus was much less efficient when raised on its natural host S. littoralis since its intrinsic growth rate turned out to be negative, which would indicate that the offspring would be lost after several generations.

Second, tests were carried out to evaluate the effect of egg age on the parasitization of C. inanitus on S. littoralis, proving that the parasitization of 48-hour-old eggs provided a higher number of females in the offspring. The effect of parasitization time on the progeny was also studied and, in this case, C. inanitus was more effective in terms of adult production by parasitizing 6 and 24 hours than at shorter times (1 and 3 hours), although no statistically significant differences were found.

Next, choice assays were performed to evaluate the ability of C. inanitus to discriminate between eggs treated or not with the specific baculoviruses of S. exigua and S. littoralis, as well as the preference between parasitizing S. exigua and S. littoralis when both species were treated with the broad-spectrum baculovirus AcMNPV. It was observed that C. inanitus preferred to attack S. exigua eggs treated with SeMNPV. However, it did not distinguish between SpliMNPV-treated or untreated eggs. No preference for any host was observed when both eggs were treated with AcMNPV.

The dispersion of the specific baculoviruses SpliMNPV and SeMNPV by C. inanitus females was also investigated, demonstrating that the parasitoid can disperse both viruses. The mortality caused by SeMNPV was much higher than that of SpliMNPV. The dispersion of baculovirus was more efficient when C. inanitus females were in contact with pepper leaves sprayed with the virus compared to eggs treated with the same virus, thus evidencing that contact is the main route of dispersion.

Finally, the parasitization preference in a S. exigua-S. littoralis multihost system and the transmission of a broad-spectrum baculovirus under these conditions were studied. Hyposoter didymator was observed to transmit AcMNPV to both lepidopterans, but its efficiency was mediated by host preference and baculovirus pathogenicity in each host. In this particular case, both H. didymator and AcMNPV showed a clear preference towards S. exigua. It was also studied whether H. didymator was capable of discriminating between S. exigua larvae infected and not infected with SeMNPV and it was shown that it equally parasitized larvae from both treatments.

RESUMEN

Spodoptera littoralis (Boisduval, 1833) y Spodoptera exigua (Hübner, 1808) son dos plagas de noctuidos cuya marcada polifagia las hace especialmente dañinas para la agricultura del sureste español, apareciendo a veces de forma individual y otras, en coexistencia. Actualmente se controlan básicamente por lucha química, dando lugar a fenómenos de resistencia, por lo que se han investigado y aplicado diversas estrategias de control, entre las que se incluye el control biológico.

Hyposoter didymator (Thunberg, 1822) y Chelonus inanitus (Linnaeus, 1767) son dos endoparasitoides koinobiontes, larvario y ovolarvario respectivamente, cuyas hembras ovipositan en el huésped, donde la larva se desarrolla y se alimenta de sus tejidos antes de tejer un capullo de seda y emerger como adulto. Ambos parasitoides pueden ser útiles para controlar especies del género Spodoptera, sobre todo S. exigua. Por otro lado, otro tipo de control biológico contra noctuidos son los baculovirus, entomopatógenos que se comercializan como insecticidas microbianos.

En la presente tesis se ha trabajado con el baculovirus de amplio espectro AcMNPV-virus de la poliedrosis nuclear de Autographa californica y los específicos de S. exigua (SeMNPV-virus de la poliedrosis nuclear de Spodoptera exigua) y S. littoralis (SpliMNPV-virus de la poliedrosis nuclear de Spodoptera littoralis), con el objetivo principal de estudiar, en condiciones de laboratorio, la dispersión de los mismos por medio de los parasitoides H. didymator y C. inanitus, analizando, además, sus interacciones.

En primer lugar, se estudió el desarrollo de C. inanitus, tanto en su huésped natural S. littoralis como en un huésped alternativo, como Ephestia kuehniella (Zeller, 1879), con la finalidad de mejorar su cría en laboratorio. Se observó que la longevidad de los adultos de C. inanitus fue significativamente más corta cuando estuvo en presencia de huevos de cada uno de los huéspedes en comparación con la ausencia de huevos. En cuanto a la reproducción, se ha demostrado que la reproducción del parasitoide C. inanitus es mucho menos eficiente cuando se cría en su huésped natural S. littoralis. ya que al calcular su tasa intrínseca de crecimiento resultó ser negativa en S. littoralis, lo cual indicaría que la cría se perdería tras varias generaciones.

En segundo lugar, se realizaron ensayos para evaluar el efecto de la edad del huevo en la parasitación de C. inanitus sobre huevos de S. littoralis, comprobándose que la parasitación de los huevos de 48 h de edad es la que proporciona un número más elevado de hembras en la descendencia. También se estudió el efecto del tiempo de parasitación sobre la progenie y, en este caso, C. inanitus es más efectivo en términos de producción de adultos parasitando 6 y 24 horas que cuando lo hace a menores tiempos (1 y 3 horas), aunque no se encontraran diferencias estadísticamente significativas.

A continuación, se realizaron ensayos de elección para evaluar la capacidad de C. inanitus de discriminar entre huevos tratados o no con los baculovirus específicos de S. exigua y S. littoralis, así como la preferencia entre parasitar S. exigua y S. littoralis cuando ambas especies han sido tratadas con el baculovirus de amplio espectro AcMNPV. Se observó que C. inanitus prefirió atacar los huevos de S. exigua tratados con SeMNPV. Sin embargo, no distinguió entre huevos tratados con SpliMNPV o no tratados y no se observó preferencia por huevos de un huésped cuando se trataron ambos plastones con AcMNPV.

También se investigó la dispersión de los baculovirus específicos SpliMNPV y SeMNPV por parte de las hembras de C. inanitus, demostrándose que el parasitoide puede dispersar ambos virus, siendo la mortalidad causada por SeMNPV mucho más elevada que la de SpliMNPV. Se comprobó que la dispersión de baculovirus fue más eficiente cuando las hembras de C. inanitus estuvieron en contacto con hojas de pimiento pulverizadas con el virus en comparación con los huevos tratados con el mismo virus, demostrándose así que el contacto es la principal via de dispersión.

Por último, se estudió la preferencia de parasitación en un sistema multihuésped S. exigua- S. littoralis y la transmisión de un baculovirus de amplio espectro en dichas condiciones. Se observó que H. didymator transmite AcMNPV a ambos lepidópteros, pero su eficiencia está mediada por la preferencia por el huésped y la patogenicidad del baculovirus en cada huésped. En este caso particular, tanto H. didymator como AcMNPV mostraron una clara preferencia hacia S. exigua. Se determinó, además, si H. didymator es capaz de discriminar entre larvas de S. exigua infectadas y no infectadas con SeMNPV y se demostró que parasita igualmente unas larvas que otras.

13 Pág., Chelonus inanitus L. (Hymenoptera: Braconidae) is an egg-larval parasitoid of Lepidoptera, with the potential for biological control of noctuids. The lack of information merits research on its reproductive and developmental biology. We studied the reproduction of mated parasitoid pairs during female lifetime and the development of the progeny on two hosts, the factitious Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) and the natural Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae). The longevity of pairs subjected or not to parasitism was compared. Development of the progeny after parasitization and growth rates were studied. Reproduction decreased the longevity of both sexes in every host. The similar survival patterns proved that sex was not a factor influencing longevity. Females parasitized immediately after emergence and offspring production peaked at 2–2.5 days. Low parasitization rates (3–12%) were generally observed. Reproduction on S. littoralis was fourfold less efficient, probably due to failure of immature stages leading to poor progeny. Sex ratio of the progeny was mostly male-biased. Males developed faster in both hosts. Development was 20 days shorter in the natural host. Strikingly, the parasitoid performed successfully better on the factitious host. Inbreeding, poor understanding of host preference, and inadequate rearing and handling practices are discussed as possible causes of the worse parasitoid performance on S. littoralis., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by MCIN/AEI/https://doi.org/10.13039/501100011033 [grants AGL2017-83498-C2-2-R, PID2020-117062RB-C22 and IJC2018-035042-I]. Ariel Morel has received fellowship from Ministerio de Hacienda de Paraguay, Programa nacional de becas de postgrado D. Carlos Antonio López [grant 01/2019]., Peer reviewed