Impairment of pupation by RNA interference-aided knockdown of Broad-Complex gene in Leptinotarsa decemlineata (Say).

Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) has a great potential for management of Leptinotarsa decemlineata. An important first step is to discover possible RNA-interference (RNAi)-target genes effective against larvae, especially the old larvae. In the present paper, five putative Broad-Complex (BrC) cDNAs (Z1-Z4, and Z6) were identified in L. decemlineata. The expression of the five LdBrC isoforms was suppressed by juvenile hormone signaling, whereas the transcription was upregulated by 20-hydroxyecdysone signaling at the fourth (final) instar larval stage. Feeding of bacterially expressed dsBrC (derived from a common fragment of the five LdBrC variants) in the third- and fourth-instar larvae successfully knocked down the target mRNAs. For the fourth-instar LdBrC RNAi hypomorphs, they had a higher larval mortality compared with the controls. Moreover, most dsBrC-fed beetles did not pupate normally. After removal of the apolysed larval cuticle, a miniature adult was found. The adult head, compound eyes, prothorax, mesothorax, metathorax were found on the dorsal view. Distinct adult cuticle pigmentation was seen on the prothorax. The mouthparts, forelegs, midlegs, and hindlegs could be observed on the ventral view of the miniature adults. For the third-instar LdBrC RNAi specimens, around 20% moribund beetles remained as prepupae and finally died. Therefore, LdBrC is among the most attractive candidate genes for RNAi to control the fourth-instar larvae in L. decemlineata.

Isoform specific roles of Broad-Complex in larval development in Leptinotarsa decemlineata.

Broad-Complex (BrC) is a downstream target of both 20-hydroxyecdysone and juvenile hormone signalling. BrC regulates morphogenetic changes between nymphal instars in hemimetabolans, whereas it controls pupal commitment, pupal morphogenesis and inhibits adult differentiation in holometabolans. Among five BrC cDNAs (Z1-Z4 and Z6) identified in the Colorado potato beetle, we found in this work that Z1, Z2 and Z6 were mainly expressed at the last (fourth) instar and prepupal stages, whereas the levels of Z3 and Z4 increased during the penultimate (third) instar stage, peaked at the last instar larval phase and gradually decreased at the prepupal and pupal periods. When knocking down all BrC isoforms by RNA interference (RNAi) at the penultimate instar stage, around 20% of the resultant larvae remained as moribund beetles. These moribund BrC RNAi larvae were completely or partially wrapped in old cuticle. Likewise, a portion of larvae treated for a single double-stranded RNA of Z3, Z4 or Z6 displayed a degree of similar aberrancies, increasing in the order of isoforms Z6 < Z3 < Z4. When silencing all BrC isoforms at the last instar period, most of the RNAi larvae did not normally pupate or emerge as adults. Separately silencing each of the five zinc finger domains revealed that approximately 70% of the Z1 RNAi larvae remained as prepupae, around 60% of the Z6 RNAi specimens formed aberrant prepupae or pupae and about 60% of the Z2 RNAi beetles became deformed pupae. After removal of the old exuviae, these deformed larvae in which either Z1, Z2 or Z6 was depleted possessed adult prothorax and mesothorax, developing antenna, mouthparts and wing discs. Moreover, less than 50% of the resultant pupae finally emerged as adults when either of Z1, Z2 or Z6 was knocked down. Therefore, our findings reveal, for the first time, that the two roles of BrC in insect groups (ie directing morphogenetic changes during juvenile development and regulating larval-pupal-adult metamorphosis) are played by different BrC isoforms in Leptinotarsa decemlineata.

Requirement of Leptinotarsa decemlineata gene within the 74EF puff for larval-pupal metamorphosis and appendage growth.

Two Drosophila melanogaster E-twenty-six domain transcription factor isoforms (E74A and E74B) act differentially at the start of the 20-hydroxyecdysone (20E) signalling cascade to regulate larval-pupal metamorphosis. In the present paper, we identified the two isoforms (LdE74A and LdE74B) in Leptinotarsa decemlineata. During the larval development stage, the mRNA transcript levels of the two LdE74 isoforms were correlated with circulating 20E titres. In vitro midgut culture and in vivo dietary supplementation with 20E revealed that the presence of 20E induced expression peaks of both LdE74A and LdE74B, with similar patterns observed for the two isoforms. Moreover, the mRNA transcript levels of both LdE74A and LdE74B isoforms were significantly downregulated in the L. decemlineata ecdysone receptor RNA interference (RNAi) specimens, but not in the LdE75 RNAi beetles. Ingestion of 20E reduced the larval fresh weights and shortened the larval development period, irrespective of knockdown of LdE74 or not. RNAi of LdE74 did not affect 20E-induced expression of the Ecdysone induced protein 75-hormone receptor 3-fushi tarazu factor 1 (E75-HR3-FTZ-F1) transcriptional cascade. Thus, it seems that LdE74 mediates 20E signalling independent of the E75-HR3-FTZ-F1 transcriptional cascade. Furthermore, silencing of both LdE74 isoforms caused failure of ecdysis. Most of the LdE74 RNAi beetles remained as prepupae. The LdE74 RNAi prepupae exhibited adult character-like forms underneath after removal of the apolysed larval cuticle. Their appendages such as antennae, legs and wings were shorter than those of control larvae. Only a few LdE74 RNAi larvae finally became deformed pupae, with shortened antennae and legs. Therefore, LdE74 is required for larval-pupal metamorphosis and appendage growth in L. decemlineata.

RNA interference-mediated functional characterization of aquaporin genes in Tribolium castaneum.

An obvious challenge faced by most terrestrial insects is maintaining water homeostasis in an arid environment. Current research suggests aquaporins may be evolved to meet the challenge. However, up to now, this suggestion has not been verified in any insect that feeds upon solid food with mandibulate mouthparts. In the present paper, nine putative aquaporin genes [Tribolium castaneum big brain, T. castaneum Drosophila integral protein (TcDrip), T. castaneum Pyrocoelia rufa integral protein (TcPrip), T. castaneum aquaporin 12-like, T. castaneum entomoglyceroporin 1 (TcEglp1), TcEglp2, TcEglp3, TcEglp4 and TcEglp5] were identified in T. castaneum. The transcripts of the nine genes were easily detectable in the foregut, midgut, hindgut-Malpighian tubule complex, fat body and carcass (except gut and fat body). Amongst them TcDrip, TcPrip, TcEglp1, TcEglp3 and TcEglp5 were highly transcribed in the hindgut-Malpighian tubule complex; TcEglp4 was abundantly expressed in both the fat body and hindgut-Malpighian tubule complex. RNA interference (RNAi)-mediated knockdown of TcEglp3 caused a grey larval cuticle, in contrast to a smooth and bright cuticle in control larvae. Approximately 40% of the TcEglp3 RNAi larvae had their hindguts protruding from the anus; their fresh wet faeces were attached to the hindgut. Another 20% of these treated larvae did not defaecate normally; wet brown faeces were adhered to the anal area. As a result, the larval growth was inhibited and about 60% larval lethality occurred. Silencing of TcEglp4 or TcDrip exhibited similar but weaker defective phenotypes as those of the TcEglp3-silenced larvae. Therefore, Eglp3, Eglp4 and Drip may contribute to the conductance of water in the alimentary canal and Malpighian tubules in T. castaneum.

Influence of probiotics in different energy and nutrient density diets on growth performance, nutrient digestibility, meat quality, and blood characteristics in growing-finishing pigs.

The study was conducted to determine the effects of probiotics (Bacillus subtilis endospore and Clostridium butyricum endospore complex) supplementation and different energy and nutrient densities on growth performance, nutrient digestibility, and meat quality in growing-finishing pigs. A total of 96 pigs with initial BW of 47.50 ± 1.14 kg were used in a 10-wk experiment. Pigs were randomly allotted to 1 of 4 treatments in a 2 × 2 factorial arrangement of treatments with 2 levels of energy and nutrient density (low and high energy, CP, and Lys) and 0.2% probiotics according to their sex and BW (6 pens, with 2 gilts and 2 barrows/pen). From 0 to 5 wk, ADG and G:F, as well as the apparent total tract digestibility (ATTD) of N and energy, were improved (P < 0.05) in the probiotic groups and in the high-energy and high-nutrient-density groups. From 6 to 10 wk, ADG and G:F, as well as the ATTD of DM, N, and energy, were increased (P < 0.01) when pigs were fed the high-energy and high-nutrient-density diets. Pigs fed the probiotic supplement or the high-energy and high-nutrient-density diets had increased (P < 0.01) ADG and G:F overall. Moreover, pigs fed the high-energy and high-nutrient-density diets had increased (P < 0.05) meat color scores, marbling scores, drip loss values, pH, and LM area (P < 0.05) compared with those fed the low-energy and low-nutrient-density diets, whereas the probiotic treatment groups had increased (P < 0.01) meat color scores, marbling scores, and redness values. The lightness and yellowness values in the low-energy and low-nutrient-density dietary treatments were greater (P < 0.05) than those in the high-energy and high-nutrient-density dietary treatments. Interactive effects of dietary energy and nutrient density and probiotics were also observed (P < 0.05) on the digestibility of N at wk 10 and energy at wk 5, as well as on meat firmness at the end of the experiment. In conclusion, dietary supplementation of probiotics increased growth performance throughout the experiment and exerted beneficial effects on ATTD. Meat quality was improved when pigs were fed high-energy and high-nutrient-density diets.