Functional characterization of a juvenile hormone esterase related gene in the moth Sesamia nonagrioides through RNA interference

Abstract

Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (SnJHER) in the corn stalk borer, Sesamia nonagrioides. SnJHER was rhythmically up-regulated close to each molt during the corn stalk borer's larval development. In this paper we attempted to functionally characterize SnJHER using several reverse genetics techniques. To functionally characterize SnJHER, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of SnJHER in the developmental programming of Sesamia nonagrioides. It is still unclear whether SnJHER is closely related to the authentic JHE gene, with different or similar biological functions.

Figure 1. Targeting the SnJHER 472/1276/1725 bp part after hemolymph injection of L5d3 and L6d9 larvae.

A. Schematic representation of SnJHER gene. The black color represents the SnJHER ORF, while the white color represents the 5′ and 3′ untranslated regions of the gene. B., E. Semiquantitative RT-PCR analysis of SnJHER mRNA levels, of a randomly selected individual injected with the dsJHER₄₇₂ as 5^th instar d3 (B.) or 6^th instar d9 larva (E.) and its randomly selected control 3 days post injection. C., F. Semiquantitative RT-PCR analysis of SnJHER mRNA levels, of a randomly selected individual injected with the dsJHER₁₂₇₆ as 5^th instar d3 (C.) or 6^th instar d9 larva (F.) and its randomly selected control 3 days post injection. D., G. Semiquantitative RT-PCR analysis of SnJHER mRNA levels, of a randomly selected individual injected with the dsJHER₁₇₂₅ as 5^th instar d3 (D.) or 6^th instar d9 larva (G.) and its randomly selected control 3 days post injection.

Figure 2. Targeting the SnJHER 472 bp part after bacterial administration of dsJHER₄₇₂.

A. Confirmation of dsJHER₄₇₂ synthesis in IPTG induced HT115 bacteria. Agarose gel electrophoresis of RNA extracted from IPTG induced HT115/L4440 (1) and HT115/pGEM T-easy-JHER_loop (2) bacteria followed by RNase-A treatment in high salinity buffer. B. Semiquantitative RT-PCR analysis of JHER gene in randomly selected pools of 10 insects recovered from a continuous feeding assay (1^st→5^h instar d0) with the HT115 bacteria (1, 2), 6 and 15 days post recovery (PR). C. Semiquantitative RT-PCR analysis of JHER, EcR, Hsp70 and Hsc70 in randomly selected pools of 10 insects from a continuous feeding assay (5^th→6^th instar) with the HT115 bacteria (1, 2), 7 days post feeding. As reference gene it was used the Sesamia’s b-tubulin gene.

Figure 3. Targeting the SnJHER 472 bp part after baculovirus administration of dsJHER₄₇₂.

A. Fluorescence field images of BmNPV-BmA::GFP/BmA::dsLuciferase and BmNPV-BmA::GFP/BmA::JHER_loop infected animals. B. Semiquantitative RT-PCR analysis of SnJHER, JHER_loop and GFP gene in randomly selected BmA::GFP/BmA::dsLuciferase (1) and BmNPV-BmA::GFP/BmA::JHER_loop infected animals (2), 7 days post infection C. Real time RT-PCR analysis of SnJHER mRNA levels in 14 randomly selected BmA::GFP/BmA::dsLuciferase (White column) and BmNPV-BmA::GFP/BmA::JHER_loop (Black column) infected individuals, 7 days post infection. The bars above the columns indicate the S.E. of the mean of 14 samples with three technical replicates.

Figure 4. Phenotypic results after hemolymph administration of dsJHERs.

A. Left: Normal Pupa, Right: Normal 6^th instar d9 larva (Prepupa). B. Targeting the SnJHER 472 bp (BI.) or the SnJHER 1276 bp (BII.) or the SnJHER 1725 bp (BIII.) part after hemolymph administration of dsJHERs in 6^th instar d9 larvae. BI. Resulted larval-pupal intermediates after hemolymph administration of dsJHER₄₇₂ in 6^th instar d9 larvae, BII. Resulted larval-pupal intermediates after hemolymph administration of dsJHER₁₂₇₆ in 6^th instar d9 larvae, BIII. Resulted larval-pupal intermediates after hemolymph administration of dsJHER₁₇₂₅ in 6^th instar d9 larvae. The arrows indicate similar abnormalities observed in intermediates of all targeted regions. In each case, -I-, -II- or -III- the abnormalities were presented in an average of >90% of the injected animals, 3 days post injection, (N = 100). C. Developmental abnormalities of dsJHER₁₇₂₅ 5^th instar d3 injected larvae. i. Normal larva, ii. Abnormal larva with fused (double) melanized epidermis of the previous instar. iii. Lateral view of abnormal larvae with fused epidermis. iv. The same larva of previous case -iii.-, by removing the melanized epidermis. The arrow indicates the fusion point between the previous and the new epidermal tissues. The newest epidermis is of yellowish colour. Scale bar: 1 cm

Figure 5. Phenotypic results after baculovirus-mediated dsJHER₄₇₂ administration.

A. Targeting the SnJHER 472 bp part after baculovirus administration of dsJHER₄₇₂ in 5^th instar d3 larvae Left. Infection with BmNPV-BmA::GFP/BmA::dsLuciferase virus 7^th day post infection (N = 200), a. Dorsal and b. Lateral view of BmNPV-BmA::GFP/BmA::dsLuciferase infected animals. Right. Infection with BmNPV-BmA::GFP/ BmA::JHER_loop virus 7^th day post infection (N = 200), 1. Type I of developmental abnormalities of BmNPV-BmA::GFP/BmA::JHER_loop infected animals, melanized epidermis in the posterior side. a. Dorsal and b. Abdominal view of BmNPV-BmA::GFP/BmA::JHER_loop infected animals. 2. Type II of developmental abnormalities of BmNPV-BmA::GFP/BmA::JHER_loop infected animals, melanized epidermis in the lateral side. a. Dorsal, b. Lateral and c. Abdominal view of BmNPV-BmA::GFP/BmA::JHER_loop infected animals. 3. Type III of developmental abnormalities of BmNPV-BmA::GFP/BmA::JHER_loop infected animals, melanized epidermis in the whole body, dorsal view. Types I, II, and III were presented in 14 % of the BmNPV-BmA::GFP/BmA::JHER_loop infected animals (N = 200). B. Targeting the SnJHER 472 bp part after baculovirus administration of dsJHER₄₇₂ in 6^th instar d9 larvae. Developmental abnormalities of larval-pupal intermediates shared in both BmNPV-BmA::GFP/BmA::dsLuciferase and BmNPV-BmA::GFP/BmA::JHER_loop infected animals (1→5). A. Normal pupa. The type 3., of larval-pupal intermediate that was presented only in BmNPV-BmA::GFP/BmA::JHER_loop infected animals. Scale bar: 1 cm

Figure 6. Targeting the SnJHER 472 bp part after baculovirus administration of dsJHER₄₇₂.

Developmental abnormalities of pupal-adult transition of BmNPV-BmA::GFP/BmA::dsLuciferase and BmNPV-BmA::GFP/BmA::JHER_loop infected animals. The scale less phenotype was presented only in the BmNPV-BmA::GFP/BmA::JHER_loop infected animals. Scale bar: 1 cm