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1.
Arch Insect Biochem Physiol ; : e21787, 2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33871104

RESUMO

High specificity for silencing target genes and single-copy target genes that yield clear phenotypes are two important factors for the success of RNA interference (RNAi). The lethal giant larvae (Lgl) gene appears to be an ideal gene for RNAi because RNAi can effectively suppress its expression and results in molting defects and mortality in Tribolium castaneum. To investigate the suitability of this gene for RNAi in other insects, we identified and characterized DvLgl from the western corn rootworm, Diabrotica virgifera virgifera, a species exhibiting high RNAi efficiency. DvLgl was expressed in all developmental stages and tissues investigated. The deduced DvLgl protein showed high amino-acid sequence identities and similar domain architecture to Lgls from other insect species. Despite many similarities among insect Lgls, RNAi-mediated suppression of DvLgl failed to produce a phenotype in D. v. virgifera adults. The difference in developing phenotypes could be attributed greatly to the level of gene suppression and the insect developmental stages for RNAi. These results highlight the variability in RNAi response among insects and showcase the importance of screening multiple target genes when conducting RNAi studies. Our findings are expected to help the design of future RNAi studies and future investigations of Lgl in insects.

2.
Arch Insect Biochem Physiol ; 106(3): e21775, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33644918

RESUMO

DDX3 represents a well-defined subfamily of DEAD-box RNA helicase and exerts multiple functions in RNA metabolism, cell cycle, tumorigenesis, signal pathway, and fertility. Our previous study has shown that LmDDX3, the ortholog of DDX3 in Locusta migratoria, is ubiquitously expressed, and with a high abundance in testis and ovary. Knockdown of LmDDX3 results in a lethal phenotype in nymph, but it still remains unclear for its role in reproductive process. In this study, we therefore characterized LmDDX3 expression in female adult locust and analyzed its function in oocyte development. LmDDX3 was expressed in all tissues examined with significant more transcripts in ovary and hindgut. In ovary, a strong expression level was detected at the day just after adult eclosion, and a dramatic reduction then occurred during the oocyte development. LmDDX3 RNAi led to a reduced vitellogenin (Vg) expression in fat body via partially at least, the JH signaling pathway, and caused an upregulation of vitellogenin receptor (VgR) in ovary, and thus blocked the ovarian development and oocyte maturation. Sequence and phylogenetic analysis indicated that LmDDX3 was closely related to termite DDX3. Taken together, these data reveal a critical role for LmDDX3 in regulating the transcription of Vg and VgR, two major factors in vitellogenesis that is a key process required for ovary development and oocyte maturation in locust, and contribute thereof a new putative target for locust biological control.


Assuntos
Locusta migratoria , Oócitos/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , RNA Helicases , Animais , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Feminino , Expressão Gênica , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Locusta migratoria/genética , Locusta migratoria/fisiologia , Ninfa/genética , Ninfa/fisiologia , Oogênese/fisiologia , Ovário/metabolismo , Filogenia , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Vitelogênese/fisiologia , Vitelogeninas/genética , Vitelogeninas/metabolismo
3.
J Insect Sci ; 21(2)2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33693804

RESUMO

Diurnal temperature amplitude is known to have a large influence on insect life history. Population density affects intraspecific competition and many other aspects of insect life history. However, there is limited information on the interactive effects of these factors on insects. Here, we tested the interactive effects of three diurnal temperature amplitudes (22 ± 0°C, 22 ± 6°C, and 22 ± 12°C) and three population densities on the development, survival, longevity, and fecundity of the English grain aphid Sitobion avenae (Fabricius) (Homoptera: Aphididae). At a constant temperature, increasing population density reduced the growth and survival of early-instar nymphs, increased longevity, and reduced fecundity. At a low population density, increasing temperature amplitude inhibited nymph development. However, even at a high temperature amplitude, nymph survival rate was higher than expected, and reproduction was possible because the recovery of the lower night-temperatures eliminated thermal stress. Increasing the population density reduced, and even reversed, the negative effects of the wide temperature amplitude. This may reflect synergistic interactions between population density and wide temperature amplitude as these stressors each incur energetic costs. These findings emphasize the importance of temperature amplitude and population density for improving prediction accuracy and damage assessment during pest control modeling.


Assuntos
Afídeos , Densidade Demográfica , Temperatura , Animais , Afídeos/crescimento & desenvolvimento , Afídeos/fisiologia , Fertilidade , Estágios do Ciclo de Vida , Ninfa/crescimento & desenvolvimento , Reprodução
4.
Insect Sci ; 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33634599

RESUMO

Halloween genes are involved in the biosynthesis of the molting hormone, which plays a key role in insect ecdysis, development, metamorphosis, and reproduction. Our previous work identified five Halloween genes from Locusta migratoria, but their functions are currently unknown. In this study, the sequences of these five Halloween genes were analyzed and characterized. LmCYP307a2, LmCYP306a1, LmCYP302a1, and LmCYP315a1 were primarily expressed in the prothoracic glands, while LmCYP314a1 was universally expressed in peripheral tissues, especially in the ovaries and Malpighian tubules. All five Halloween genes were mainly expressed from the 5th to the 7th d in 5th-instar nymphs. RNA interference (RNAi) silencing of LmCYP307a2 resulted in severe molting delays and molting failure, which could be rescued by supplementary 20-hydroxyecdysone. A hematoxylin and eosin staining analysis suggested that the RNAi of LmCYP307a2 inhibited the ecdysis process by inhibiting the apolysis and degradation of the old cuticle, and by promoting the synthesis of a new cuticle. Quantitative reverse transcription polymerase chain reaction results showed that the expressions of LmE74, LmCht5, and LmCht10 were dramatically down-regulated, while that of LmChsI was substantially up-regulated, after knockdown of LmCYP307a2. The results suggest that LmCYP307a2 is related to the molt process via regulation of chitin synthesis and degradation.

5.
J Insect Physiol ; 129: 104181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33359365

RESUMO

RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.

6.
Insect Sci ; 2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33140888

RESUMO

The efficiency of RNA interference (RNAi) varies substantially among different insect species. Rapid degradation of double-stranded RNA (dsRNA) by dsRNA-degrading nucleases (dsRNases) has been implicated to cause low RNAi efficiency in several insect species. In this study, we identified four dsRNase genes (OfdsRNase1, OfdsRNase2, OfdsRNase3 and OfdsRNase4) from the Asian corn borer (Ostrinia furnacalis) transcriptome database. Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides. Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae. RNAi efficiency was investigated in 2-d-old fifth-instar larvae (high expression of dsRNase2) and 2-d-old pupae (low expression of dsRNase2) by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein (OfLgl). Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae, but not in larvae, suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages. This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene, OfHex1, was significantly improved after knockdown of OfdsRNase2. When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro, only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA. Taken together, our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O. furnacalis larvae.

7.
Pest Manag Sci ; 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002336

RESUMO

BACKGROUND: Targeting insect-specific genes through post-transcriptional gene silencing with RNA interference (RNAi) is a new strategy for insect pest management. However, lepidopterans are recalcitrant to RNAi, which prevents application of novel RNAi technology to many notorious pests, including Ostrinia nubilalis (ECB). Strategies for enhancing RNAi efficiency, including large doses of double-stranded RNA (dsRNA), nuclease inhibitors, transfection reagents, and nanoparticles, have proved useful in other insects exhibiting substantial dsRNA degradation, a major mechanism limiting RNAi efficacy. To determine if similar strategies can enhance RNAi efficiency in ECB, various reagents were tested for their ability to enhance dsRNA stability in ECB tissues, then compared for their effectiveness in whole ECB. RESULTS: Ex vivo incubation experiments revealed that Meta dsRNA lipoplexes, EDTA, chitosan-based dsRNA nanoparticles, and Zn2+ enhanced dsRNA stability in ECB hemolymph and gut content extracts, compared with uncoated dsRNA. Despite these positive results, the reagents used in this study were ineffective at enhancing RNAi efficiency in ECB in vivo. To reduce assay time and required dsRNA, midguts were dissected and incubated in tissue culture medium containing dsRNA with and without reagents. These experiments showed that RNAi efficiency varied between target genes, and nuclease inhibitors improved RNAi efficiency for only a portion of the refractory target genes investigated ex vivo. CONCLUSION: These results indicate that enhancing dsRNA stability is insufficient to improve RNAi efficiency in ECB and suggests the existence of additional, complex mechanisms contributing to low RNAi efficiency in ECB.

8.
Insect Sci ; 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-33037856

RESUMO

Chitin deacetylases (CDAs, including CDA1 and CDA2) are considered key enzymes for body cuticle formation and tracheal morphogenesis in various insect species. However, their functions in the formation of the cuticular intima of the foregut and hindgut are unclear. Here, we investigated the roles of their respective genes LmCDA1 and LmCDA2 in this process, in the hemimetabolous insect Locusta migratoria. Transcripts of LmCDA1 and LmCDA2 were highly expressed both before and after molting in the foregut. In the hindgut, their expression was high only before molting. In both the foregut and hindgut, LmCDA1 protein was localized in the basal half of the chitin matrix (procuticle), whereas LmCDA2 was detected in the upper half of the procuticle. Knockdown of LmCDA1 by RNA interference (RNAi) in 5th-instar nymphs caused no visible defects of the hindgut cuticle. By contrast, the chitinous lamellae of the cuticular intima in the foregut of knockdown animals were less compact than in control animals. RNAi against LmCDA2 led to thickening of both the foregut and hindgut cuticles, with a greater number of thinner laminae than in the respective control cuticles. Taken together, our results show that LmCDA1 and LmCDA2 have distinct, but overlapping, functions in chitin organization in the foregut cuticle. However, in the hindgut, this process seems independent of LmCDA1 activity but requires LmCDA2 function. Thus, the CDAs reflect tissue-specific differences in cuticular organization and function, which need further detailed molecular and histological analyses for full comprehension.

9.
Pestic Biochem Physiol ; 170: 104700, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980067

RESUMO

Argonautes (Ago) are important core proteins in RNA interference (RNAi) pathways of eukaryotic cells. Generally, it is thought that Ago1, Ago2 and Ago3 are involved in the miRNA (microRNA), siRNA (small interfering RNA) and piRNA (Piwi-interacting RNA)-mediated RNAi pathways, respectively. As a main component of the RNA-induced silencing complex (RISC), Ago2 plays an indispensable role in using siRNA to recognize and cut target messenger RNAs resulting in suppression of transcript levels, but the contributions of Ago1 and Ago3 to the siRNA-mediated RNAi pathway remain to be explored in many insect species. In this study, we investigated the contributions of four Ago genes (named LmAgo1, LmAgo2a and LmAgo2b and LmAgo3) to RNAi efficiency in Locusta migratoria by using both in vivo and in vitro experiments. Our results showed that suppression of each of the Ago genes significantly impaired RNAi efficiency when targeting Lmß-tubulin transcripts, resulting in recovery of 48, 43.3, 61.4 or 26% of Lmß-tubulin transcripts following RNAi-mediated suppression of LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3, respectively. Furthermore, overexpression of LmAgo1, LmAgo2a, LmAgo2b, or LmAgo3 in a PAc5.1-V5/HisB vector and co-transfection with psicheck2 fluorescence vector in S2 cells reduced luciferase fluorescence by 38.3, 58.9, 53.3 or 55.6%, respectively. Taken together, our results showed that LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3 each make significant contributions to RNAi efficiency in L. migratoria and suggest that the involvement of all four enzymes could be one of the major factors supporting robust RNAi responses observed in this species.


Assuntos
Locusta migratoria/genética , MicroRNAs/genética , Animais , Proteínas Argonauta/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética
10.
Insects ; 11(10)2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32977554

RESUMO

Variable RNA interference (RNAi) efficiencies limit RNAi-based pest management strategies for many pests. Previous efforts to understand mechanisms contributing to low RNAi efficiency indicate that double-stranded RNA (dsRNA) is degraded in the European corn borer (ECB), Ostrinia nubilalis, due to nuclease activity. To investigate the contribution of dsRNA-degrading endonucleases (dsRNases) and lepidopteran-specific RNAi efficiency-related nucleases (REases) to dsRNA instability and low RNAi efficiency in ECB, five complementary DNAs putatively encoding four dsRNases (OndsRNase1, 2, 3, and 4) and one REase (OnREase) were sequenced. Characterization of these transcripts revealed that substrate specificity might vary among the four dsRNases due to different amino acid combinations in the substrate-binding sites. Gene expression analysis indicated that OndsRNase2 and OnREase were highly expressed in the larval gut, and OndsRNase1 showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of OnREase rapidly increased upon dsRNA ingestion or injection, whereas OndsRNase4 expression only increased after long-term ingestion of dsRNA. While the biological function of these nucleases remains to be verified, our results suggest that OnREase and OndsRNase2, and OndsRNase1 and OndsRNase4 may be responsible for degradation of dsRNAs in the ECB gut and hemolymph, respectively, thereby contributing to low RNAi efficiency.

11.
Front Physiol ; 11: 790, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32733279

RESUMO

Apolipophorins are carrier proteins that bind lipids and mediate their transport from tissue to tissue in animals. Apolipophorin I and II (apoLp-II/I) are the major apolipophorins in insects. The implication of apoLp-II/I in cuticle lipid-barrier formation in insects has not been addressed to date. In the present study, we investigated the function of apoLp-II/I in the migratory locust Locusta migratoria (LmapoLp-II/I). During the development of fifth instar nymphs, LmapoLp-II/I transcript levels increased until mid-instar, and then decreased gradually until molting to the adult stage. We found that LmapoLp-II/I was predominately expressed in the fat body and the integument including oenocytes and epidermal cells. Immunodetection experiments revealed that LmapoLp-I mainly localized in the cytoplasm of oenocytes and epidermal cells. Silencing of LmapoLp-II/I caused molting defects in nymphs. Importantly, RNA interference against LmapoLp-II/I resulted in a significant decrease in the content of cuticle surface lipids including alkanes and methyl alkanes. Cuticular permeability was significantly enhanced in these nymphs in Eosin Y penetration assays. By consequence, desiccation resistance and insecticide tolerance of dsLmapoLp-II/I-treated locusts were reduced. Taken together, our results indicate that LmapoLp-II/I is involved in the transport and deposition of surface-cuticular lipids that are crucial for maintaining normal cuticle barrier function in L. migratoria.

12.
Pestic Biochem Physiol ; 169: 104672, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32828377

RESUMO

RNA interference (RNAi) is a revolutionary technique for silencing gene expression, but the success of this technique is dependent upon the stability of double-stranded RNA (dsRNA) molecules. In many insects, especially lepidopteran species, RNAi efficiency is limited by high instability of dsRNA in the gut and/or hemolymph, preventing the development of RNAi-based strategies for many serious pests. Previous attempts to perform RNAi on Ostrinia nubilalis (ECB, Lepidoptera: Crambidae) indicate low RNAi efficiency with both dsRNA injection and feeding. To investigate the contribution of dsRNA instability to low RNAi efficiency in ECB, a serious of ex vivo incubation experiments were performed where dsRNA integrity was assessed following incubation in larval gut continents and hemolymph using gel electrophoresis or RT-qPCR. DsRNA was less stable in the gut contents from ECB than in gut contents from Diabrotica virgifera virgifera, a coleopteran exhibiting high RNAi efficiency. Furthermore, characterization of dsRNA stability in ECB gut contents and hemolymph revealed that dsRNA was rapidly degraded under physiologically relevant conditions as a result of enzymatic activity that was neither size- nor sequence-dependent. These findings suggest that instability of dsRNA in ECB tissues is a contributing factor to the poor efficiency of RNAi in this pest. This work advances our understanding of mechanisms impacting RNAi efficiency in ECB and related lepidopteran insects for which novel pest management strategies are needed, and may facilitate the development of strategies for enhancing dsRNA stability in ECB tissues.


Assuntos
Microbioma Gastrointestinal , RNA de Cadeia Dupla , Animais , Hemolinfa , Larva , Interferência de RNA
13.
Pestic Biochem Physiol ; 168: 104637, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32711771

RESUMO

Cytochrome P450 monooxygenases (CYPs) serve many functions in insects, from the regulation of development to xenobiotic detoxification. Several conserved CYPs have been shown to play a role in insect growth and development. CYP303A1 is a highly conserved CYP with a single ortholog in most insects, but its underlying molecular characteristics and specific physiological functions remain poorly understood. In Drosophila melanogaster and Locusta migratoria, CYP303A1 is indispensable for eclosion to adult. Here, we report additional functions of the locust gene LmCYP303A1 in nymphal molts, cuticular lipid deposition and insecticide penetration. RT-qPCR revealed that LmCYP303A1 had a high expression level before ecdysis and was highly expressed in integument, wing pads, foregut and hindgut. Suppression of LmCYP303A1 expression by RNA interference (RNAi) caused a lethal phenotype with molting defect from nymph to nymph. In addition, LmCYP303A1 RNAi resulted in locusts being more susceptible to desiccation and to insecticide toxicity. Furthermore, knockdown of LmCYP303A1 efficiently suppressed the transcript level of key genes (ELO7, FAR15 and CYP4G102) responsible for cuticular hydrocarbon (CHC) synthesis, which led to a decrease in some CHC levels. Taken together, our results suggest that one of the functions of LmCYP303A1 is to regulate the biosynthesis of CHC, which plays critical roles in protecting locusts from water loss and insecticide penetration.


Assuntos
Inseticidas , Locusta migratoria , Animais , Dessecação , Drosophila melanogaster , Hidrocarbonetos , Proteínas de Insetos
14.
Nephrology (Carlton) ; 25(11): 839-844, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32536031

RESUMO

AIM: Cardiovascular events (CVE) are common co-morbidities amongst patients with chronic kidney disease (CKD). The impact of CVE on the subsequent pattern and rate of deterioration of kidney function is not well described. METHODS: A retrospective cohort study of 1123 Royal Brisbane and Women's Hospital patients enrolled in the CKD.QLD registry from May 2011 to August 2017 was undertaken. Participants CVE data and renal function (eGFR CKD-EPI) were extracted from clinical records. Participants who ultimately started kidney replacement therapy (KRT) were imputed an eGFR of 8 mL/min/1.73 m2 at the date of the first KRT treatment. Annualized percentage delta eGFR was used to explore the association between CVE and rate of renal deterioration. Mortality was ascertained through electronic health records. RESULTS: There were 235 CVE events amongst 222 participants over a period of 6 years. One hundred and forty-four participants experienced ischaemic heart disease (IHD), 51 participants had stroke, 40 participants had peripheral vascular disease (PVD) and 13 participants had more than one event. CVE were associated with significantly shorter time to death in participants who experienced one CVE compared with those without a CVE (1901.2 days vs 2259 days [P < .05]). However, there was no significant change in the absolute mean delta eGFR between participants with CVE and without CVE after adjustment for age (3.8 mL/min/1.73 m2 vs 3.8 mL/min/1.73 m2 [P = .9]). Furthermore, there was no significant difference in the progression to KRT in participants with CVE compared with participants without CVE (1315 days and 1052 days (P = .46). CONCLUSION: Cardiovascular events are associated with increased mortality in the CKD cohort. They were not associated with accelerated deterioration of kidney function.

15.
Insect Sci ; 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32524775

RESUMO

Cytochrome P450 monooxygenases (CYPs) play essential physiological functions in insects. CYP303A1 is highly conserved in insect species studied to date, and shows an indispensable role for adult eclosion in both Locusta migratoria and Drosophila melanogaster. However, how CYP303A1 is regulated to control insect developmental processes remains uninvestigated. In this study, we discovered functional binding sites for miR-184 in the coding sequence of LmCYP303A1. The luciferase reporter assay showed that miR-184 could target LmCYP303A1 and regulate its expression in vitro. Furthermore, overexpression of miR-184 through microinjection of agomir to locusts reduced the transcripts of LmCYP303A1 and led to abnormal molting, which is similar to the phenotype of silencing LmCYP303A1 by direct injection of dsLmCYP303A1 to locusts. Meanwhile, down-regulation of miR-184 by injection of antagomir increased the LmCYP303A1 transcript and caused molting defects. These findings suggested that miR-184 could target LmCYP303A1 to regulate the molting process in L. migratoria, which might be considered as a novel target for pest control.

16.
Pest Manag Sci ; 76(9): 2907-2917, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32358831

RESUMO

BACKGROUND: Knickkopf (Knk) proteins play crucial roles in the formation of insect cuticle. Recent studies in the holometabolous insect red flour beetle (Tribolium castaneum) have shown that three Knk genes encoding TcKnk, TcKnk2 and TcKnk3 play different but essential roles at different developmental stages and in different tissues. However, the functions of Knk genes had not been fully explored in hemimetabolous insects such as the migratory locust Locusta migratoria. RESULTS: We identified three transcripts of LmKnk-like genes LmKnk2 and LmKnk3 with the full-length cDNA sequences, which were named as LmKnk2, LmKnk3-FL and LmKnk3-5'. These three transcripts were highly expressed before molting and mainly expressed in the integument. Among these genes, silencing only LmKnk3-5' by RNA interference (RNAi) caused molting defects and high mortality of the locusts. Injection of dsLmKnk3-5' dramatically decreased chitin content, but did not affect cuticle laminar ultra-structures in the integument. After the knockdown of LmKnk3-5' transcript, lipid deposition on the cuticle surface was impeded, and locusts exhibited increased susceptibility to each of four insecticides in three different classes. However, no visible phenotypic changes were observed after LmKnk2 or LmKnk3-FL was silenced by RNAi. CONCLUSION: We demonstrate that LmKnk3-5' is essential for cuticle formation in L. migratoria. This contrasts the findings that the cognate protein in T. castaneum TcKnk3-5' is dispensable for cuticle formation and survival. Hence, we provide some evidence that the function of Knk-type proteins may be species-specific. We therefore think that LmKnk3-5' may be a good target for the application of RNAi-based technologies for species-specific insect pest management. © 2020 Society of Chemical Industry.

17.
Pest Manag Sci ; 76(11): 3541-3550, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32419293

RESUMO

BACKGROUND: Cuticular hydrocarbons (CHCs) have a critical role in preventing desiccation and penetration of xenobiotics in insects. Previous studies have shown that cytochrome P450 subfamily 4G (CYP4G) enzymes are oxidative decarbonylases, essential for CHC biosynthesis. However, it is unclear whether there are functional differences between the two CYP4G genes in most insects. In Locusta migratoria, we identified two CYP4G genes (LmCYP4G62 and LmCYP4G102). LmCYP4G102 plays a critical role in the synthesis of CHCs, but the function of LmCYP4G62 is unknown. RESULTS: We identified, characterized, and compared two LmCYP4G genes, based on L. migratoria transcriptomic and genomic databases. RT-qPCR showed that both were highly expressed in tissues with which oenocytes are associated, the integument and fat body. Immunostaining indicated that LmCYP4G62 and LmCYP4G102 were highly abundant in oenocytes in these tissues. However, the two enzymes had a different subcellular distribution, with LmCYP4G62 localized on the plasma membrane and LmCYP4G102 dispersed throughout the oenocyte cytoplasm, presumably on the endoplasmic reticulum. RNA interference-mediated gene silencing against each of the two genes resulted in reduced CHC contents, in all classes for LmCYP4G102, but mostly shorter chain CHCs for LmCYP4G62. Silencing of both genes resulted in increased insecticide penetration through the cuticle, and increased locust susceptibility to desiccation and insecticides. CONCLUSION: Our studies suggest that both LmCYP4G62 and LmCYP4G102 contribute to hydrocarbon biosynthesis and play key roles in protecting locusts from water loss and insecticide penetration, but they are not fully redundant. Further, the two LmCYP4G genes might be used as new targets for insect pest management. © 2020 Society of Chemical Industry.


Assuntos
Locusta migratoria , Animais , Sistema Enzimático do Citocromo P-450 , Proteínas de Insetos/genética , Insetos , Inseticidas , Tegumento Comum , Locusta migratoria/genética
18.
Insect Biochem Mol Biol ; 123: 103404, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32428561

RESUMO

Mucins are highly glycosylated proteins that are characterized by a higher proportion of threonine, serine, and proline residues in their sequences. Although mucins in humans and vertebrates have been implicated in many biological processes, their roles in growth and development in invertebrates such as in insects remain largely unknown. Based on bioinformatic analyses, we identified eight mucin or mucin-like genes in the migratory locust, Locusta migratoria. RNA interference against these genes demonstrated that three Lmmucin genes were essential for the survival of L. migratoria nymphs, and one Lmmucin was required for adult wing development. Indeed, knockdown of Lmhemomucin and Lmmucin-12 caused lethal phenotypes, with an observed defect of the gastric caeca in which cells were detached from cell junctions. Deficiency of LmIIM3 resulted in lethality of nymphs, with defects of the peritrophic membrane in midgut. Suppression of Lmmucin-17 greatly impaired the structural integrity of the wing cuticle during nymph-adult molting. The present study revealed the significance of mucin and mucin-like genes in insect growth and development, using the orthopteran insect locust as a model.

19.
Insect Sci ; 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32401389

RESUMO

Wings are an important flight organ of insects and their morphogenesis depends on a series of cell-to-cell and cell-to-extracellular matrix interactions. Integrin as a transmembrane protein receptor mediates cell-to-cell adhesion, cell-to-extracellular matrix interactions and signal transduction. In the present study, we characterized an integrin gene that encodes integrinß-PS protein in Locusta migratoria. LmIntegrinß-PS is highly expressed in the wing pads and the middle stages of 5th instar nymphs. Immunohistochemical analysis revealed that the LmIntegrinß-PS protein was localized at the cell base of the two layers of wings. After suppression of LmIntegrinß-PS by RNA interference, the wing pads or wings were unable to form normally, with a blister wing appearance during nymph to nymph transition and nymph to adult transition. We further found that the dorsal and ventral epidermis of the wings after dsLmIntegrinß-PS injection were improperly connected and formed huge cavities revealed by hematoxylin and eosin staining. Furthermore, the morphology and structure of the wing cuticle was significantly disturbed which affected the stable arrangement and attachments of the wing epidermis. Moreover, the expression of related cell adhesion genes was significantly decreased in LmIntegrinß-PS-suppressed L. migratoria, suggesting that LmIntegrinß-PS is required for the morphogenesis and development of wings during molting by stabilizing cell adhesion and maintaining the cytoskeleton of these cells.

20.
J Insect Physiol ; 123: 104052, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32259526

RESUMO

Insect cuticular lipids are a complex cocktail of highly diverse cuticular hydrocarbons (CHCs), which form a hydrophobic surface coat to maintain water balance and to prevent desiccation and penetration of exogenous substances. Fatty acid elongases (ELOs) are key enzymes that participate in a common CHC synthesis pathway in insects. However, the importance of ELOs for CHC synthesis and function remains understudied. Using transcriptomic data, we have identified seven ELO genes (LmELO1-7) in the migratory locust Locusta migratoria. We determined their tissue-specific and temporal expression profiles in fifth instar nymphs. As we are interested in cuticle barrier formation, we performed RNA interference against LmELO7, which is mainly expressed in the integument. Suppression of LmELO7 significantly decreased its expression and caused lethality during or shortly after molting. CHC quantification by GC-MS analysis indicated that suppression of LmELO7 resulted in a decrease in total CHC amounts. By consequence, CHC deficiency reduced desiccation resistance and enhanced cuticle permeability in LmELO7-suppressed L. migratoria. Interestingly, LmELO7 expression is induced at low air humidity. Our results indicate that LmELO7 plays a vital role in the production of CHCs and, hence, cuticle permeability. Induction of LmELO7 expression in drought conditions suggests a key role of this gene in regulating desiccation resistance. This work is expected to help developing new strategies for insect pest management based on CHC function.

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