INFLUÊNCIA DA IDADE DE OVOS DE TRICHOPLUSIA NI (LEPIDOPTERA: NOCTUIDAE) EM FÊMEAS DE TRICHOGRAMMA PRETIOSUM (HYMENOPTERA: TRICHOGRAMMATIDAE) EM DIFERENTES TEMPERATURAS

Objetivamos avaliar a influência da idade dos ovos de Trichoplusia ni (Lepidoptera: Noctuidae) em fêmeas de Trichogramma pretiosum (Hymenoptera: Trichogrammatidae), em diferentes temperaturas. Assim, ovos de T. ni com idade ≤ 12, ≤ 24, ≤ 36, ≤ 48, ≤ 60 e ≤ 72 horas de desenvolvimento embrionário, separados a temperaturas de 20, 25 e 30 ºC, foram oferecidos para fêmeas de T. pretiosum com até 24 horas de idade. O parasitismo foi inversamente proporcional ao desenvolvimento embrionário do ovo, com maiores taxas de parasitismo observadas para ovos com até 24 horas de desenvolvimento embrionário nas três temperaturas. A viabilidade do parasitismo foi influenciada pela idade dos ovos. Os ovos, com até 36 horas de idade, apresentaram viabilidade superior a 85% nas três temperaturas. A proporção sexual a 25ºC apresentou a melhor taxa dentro da faixa de desenvolvimento embrionário. O número de descendentes do parasitoide por ovo foi influenciado pela temperatura e pela idade dos ovos, sendo a combinação ovos com 60-72 horas à temperatura de 30ºC, a que apresentou o maior quantitativo de descendentes parasitoides por ovo. Esses resultados indicaram que a idade do hospedeiro e a temperatura ambiente podem alterar as características biológicas dos parasitoides.

The potential application of Trichoplusia ni granulovirus En3 enhancin as a synergist in baculovirus-based insecticides

The increased costs associated with baculovirus mass-production urge the search for synergistic products that reduce the amount of active matter. In the present thesis, a synergistic factor with great potential for baculovirus-based formulations was expressed and produced using a baculovirus expression system. The main achievement of the present thesis is that the in vivo production of solubilized enhancins using baculovirus-based expression systems can be used to improve the efficacy of biological insecticides against lepidopteran pests, reducing the active matter of bioinsecticides and making them commercially competitive with chemicals.

Assessing the impact of a viral infection on the expression of transposable elements in the cabbage looper moth (Trichoplusia ni)

Most studies of stress-induced transposable element (TE) expression have so far focused on abiotic sources of stress. Here we analyzed the impact of an infection by the AcMNPV baculovirus on TE expression in a cell line (Tnms42) and midgut tissues of the cabbage looper moth (Trichoplusia ni). We find that a large fraction of TE families (576/636 in Tnms42 cells and 503/612 in midgut) is lowly expressed or not expressed at all (≤ 4 Transcripts Per Million [TPM]) in the uninfected condition (median TPM of 0.37 in Tnms42 and 0.46 in midgut cells). In the infected condition, a total of 62 and 187 TE families were differentially expressed (DE) in midgut and Tnms42 cells, respectively, with more up- (46) than down- (16) regulated TE families in the former and as many up- (91) as down- (96) regulated TEs in the latter. Expression log2 fold changes of DE TE families varied from -4.95 to 9.11 in Tnms42 cells, and from -4.28 to 7.66 in midgut. Large variations in expression profiles of DE TEs were observed depending on the type of cells and on time after infection. Overall, the impact of AcMNPV on TE expression in T. ni is moderate, but potentially sufficient to affect TE activity and genome architecture. Interestingly, one host-derived TE integrated into AcMNPV genomes is highly expressed in infected Tnms42 cells. This result shows that virus-borne TEs can be expressed, further suggesting that they may be able to transpose, and that viruses may act as vectors of horizontal transfer of TEs in insects.

Extended in vivo transcriptomes of two ascoviruses with different tissue tropisms reveal alternative mechanisms for enhancing virus reproduction in hemolymph

Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.

A recombinant baculovirus TnGV in Trichoplusia ni larvae using the PIG bombardment method and the CRISPR/Cas9 system

Baculoviruses have been used for the expression of heterologous proteins of biotechnological interest. However, most of these proteins are obtained by homologous co-transfection recombination in cell lines, limiting their use. Recently, the CRISPR/Cas9 system has excelled in its high efficiency in editing specific sequences without the need for insect cell lines. In this work, the CRISPR/Cas9 system was used to edit the genome of Trichopusia ni granulovirus (TnGV) and transformation of insects by the PIG bombardment method. A homologous repair vector (pTnGV101) was designed with regions orf5 and orf7, as well as sgRNA flanking TnGV P10 of this virus. The bombardment transformation was carried out at 175 psi with 40% of infected T. ni larvae, of which 38% expressed the reporter protein EGFP. These results demonstrate that the CRISPR/Cas9 system and PIG bombardment can be used for genetic modification of baculovirus in vivo.

Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutants of Tyrosinase

Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene (TYR) is mutated in many cases of oculocutaneous albinism type phenotype in patients with OCA1 have only began to be examined and remain to be delineated. Here, we analyze the temperature-dependent kinetics of wild-type Tyr (WT) and two OCA1B mutant variants (R422Q and P406L) using Michaelis–Menten and Van’t Hoff analyses. Recombinant truncated human Tyr proteins (residues 19–469) were produced in the whole insect Trichoplusia Ni larvae. Proteins were purified by a combination of affinity and size-exclusion chromatography. The temperature dependence of diphenol oxidase protein activities and kinetic parameters were measured by dopachrome absorption. Using the same experimental conditions, computational simulations were performed to assess the temperature-dependent association of L-DOPA and Tyr. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L followed by dopachrome formation is a complex reaction supported by enthalpy and entropy forces. We show that the WT has a higher turnover number as compared with both R422Q and P406L. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity and to discover novel therapies for patients.