Two vital genes, ECR and RPL19 of B. dorsalis, namely were selected and their potential role in normal growth, development, and metamorphosis in different developmental stages of B. dorsalis were accomplished by ingestion from bacterial expressed corresponding dsRNA. To reduce the cost of production, increase the stability of dsRNA, and for ease of treatments to the maggot, bacteria expressing dsRNA was used instead of dsRNA extract. Remarkably, we found that dsRNA-mediated gene silencing effect through oral administration of bacteria was persistent and effective as demonstrated by reduced survival of maggots, pupae, and adult fruit flies and deformities phenotypically observed especially in pupae and adults stages.
Amplification of candidate genes fragments and sequence submission
Based on NCBI database search, different available sequences of ECR (JX105044.1, JQ034623.1) and RPL19 (HQ315780.1, KJ028105.1) genes of B. dorsalis were retrieved, and their conserved domain search was attempted to design the specific primers. The 692bp ECR and 355bp RPL19 genes fragments were amplified using gene-specific primer sets (Fig. 2a, b). This partial cDNA sequence of ECR shared 99% identity in sequence alignment with B. dorsalis, 94% sequence identity to Ceratitis capitata, 85% identity to Calliphora vicina, and 84% identity to Lucilia cuprina. The partial cDNA sequence of RPL19 shared 97% identity to B. dorsalis and 93% identity to B. minax. These isolated ECR and RPL19 gene fragments sequences were submitted on GenBank with the accession number MN311494.1 and MN311495.1, respectively.
Endogenous expression of ECR and RPL19 genes of B. dorsalis in maggot and adult stages
Total RNA was isolated from maggot and adult B. dorsalis reared on guava fruits and cDNA was synthesized using its equal concentration. Actin gene-specific amplification was chosen as an internal control because of its stability in expression during all the insect stages and its expression is less likely to be affected by environmental condition (Shen et al. 2010). Endogenous transcript expression of target gene is always important before attempting RNAi studies. To investigate the endogenous transcript expression of ecr and rpl19 genes in maggot as well as adult stages of B. dorsalis, equal amount of cDNA as template was used in reverse transcription-PCR using genes-specific primers. The transcript expression of ecr was higher in maggot as compared to adult fruit fly stage (Fig. 2b, c). On the other hand, transcript expression of rpl19 was nearly the same in both maggot and adult stages of fruit fly (Fig. 2b, c). Further, relative transcript expression of rpl19 was much higher than that of ecr gene (Fig. 2b, c).
Effects of feeding bacteria expressing dsRNA of Bdecr on B. dorsalis mortality and its ECR gene expression
In order to express dsRNA, ECR gene fragment was cloned in pL4440 RNAi feeding vector plasmid. Upon induction in IPTG, corresponding dsRNA was produced in HT115 (DE3) bacterial strain harboring pL4440-ECR construct (Fig. 2d). Bacteria expressing dsRNA-ecr feeding to maggots of B. dorsalis experiment was attempted in 2 consecutive years (2018-19). In both years, the mortality/deformity rate of maggots, pupae, and adult emerged were increased with the increase in concentration of bacteria expressing dsRNA of Bdecr in artificial diet. Seven hundred microlitre concentration of bacteria produced highest mortality and deformities as compared to that of 350 μl and 200 μl of bacterial concentration (Fig. 3a). In 700 μl bacterial treatment, 56.7% mortality of maggots along with deformities in pupal and adult stages within 15 days of start of feeding (Fig. 3) were observed, followed by 32.9 and 16.7% in 350 μl and 200 μl, respectively, on an average data of the 2 years experiments. Of the pupae developed from survived treated maggots, 51, 24.9, and 14% mortality was observed in 700, 350, and 200 μl bacterial treatments respectively in the 2 consecutive years. Of the emerged adults from survived pupae, mortality of 35.3, 19, and 10.5% were observed in 700, 350, and 200 μl bacterial treatments respectively in the 2 consecutive years (Fig. 3, Supplementary data Table 1). Finally very few adult fruit flies were survived at the end. On comparing 2 years data, the mortality of maggots, pupae, and adults was higher in 2018 than in 2019. Thus, total mortality/deformity of maggots, pupae, and adult fruit flies was 86.3% in 2018–2019 and was highest in 700 μl bacterial treatment compared to 55 and 35.8% in 350 and 200 μl dsRNAs Bdecr bacterial treatments, respectively (Supplementary data Table 1).
Thus, it appears that ecr-dsRNA was more effective to trigger RNAi silencing at early developmental stages of fruit fly. Present results were consistent with the observed RNAi silencing effect as in Nilaparveta lugens, where feeding of dsRNA targeting ecr and usp genes to nymphs resulted in disruption of molting and reduced the survival of nymphs (Yu et al. 2014). Earlier, RNAi targeting ecr and usp genes injected into larvae of Bombyx mori resulted in 40% pre-pupal death and failure to undergo larval-pupal metamorphosis and stopped at larval-pupal intermediates. Feeding of ecr-dsRNA through artificial diet to second instar grain aphids (Sitobion avenae) resulted in 50% aphid mortality after 4 days of treatment (Yan et al. 2016). The role of ecr is significantly important during the immature stages and its endogenous expression was higher in maggot stage than in adult stage of B. dorsalis. And maggots showed significant and higher mortality and deformity upon ingestion of desired dsRNA, which was a proof of high gene silencing efficiency (Fig. 3a–c). Since the requirement of rpl19 gene product is very common and important during all growth developmental stages of every insect pest and thus, required throughout the life cycle. Thus, obviously the endogenous expression of rpl19 was nearly same in both maggot and adult stages. Therefore, both maggot and adult stages of B. dorsalis showed significant mortality and deformity (Fig. 3a–c).
Gene silencing in terms of reduced relative transcript level of endogenous BdECR gene was also observed after 15 days of feeding bacteria (700 μl) expressing dsRNA-ECR in treated third instar maggots and subsequent emerged adult fruit flies which was reduced by 33 and 23%, respectively, than the native ECR gene expression in B. dorsalis (Fig. 3c).
Effects of feeding bacteria expressing dsRNAs of Bdrpl19 on B. dorsalis mortality and its RPL19 gene expression
In order to express dsRNA, RPL19 gene fragment was also cloned in pL4440 RNAi feeding vector plasmid. Upon induction in IPTG, corresponding dsRNA was produced in HT115 (DE3) bacterial strain harboring pL4440-RPL19 construct (Fig. 2d). Bacteria expressing dsRNA-rpl19 feeding to maggots of B. dorsalis experiment was also attempted in the 2 consecutive years (2018–2019). In both years, the mortality (including deformity) rate was increased with increase in concentration of bacteria expressing dsRNA as was in case of ecr. Seven hundred microliter concentration of bacteria produced higher mortality and deformities as compared to that of 350 μl and 200 μl (Fig. 3b, Supplementary data Table 2). In 700 μl treatment, 27.9% mortality of maggots was observed, followed by 19.2 and 7.5% in 350 and 200 μl, respectively, on an average data of the 2 years (2018–2019) experiments. Of the pupae developed, 45.5, 27.8, and 18.5% mortality was observed in 700, 350, and 200 μl treatments, respectively, and of the emerged adults, mortality of 69.8% (along with deformities in maggot and pupal stages) (Fig. 3), 44.3, and 26% was observed in 700, 350, and 200 μl treatments, respectively in 2018–2019. On comparing the 2 years data, the mortality rate of maggots and adults was little higher in 2019 than in 2018. Thus, on an average, total mortality/deformity of maggots, pupae, and adult fruit flies was 87.9% in 2018–2019 and was highest in 700 μl dsRNAs of Bdrpl19 bacterial treatment compared to 67.5 and 44.2% in 350 and 200 μl bacterial treatments, respectively (Supplementary data Table 2). Feeding of bacteria expressing rpl19-dsRNA at 700 μl dose to maggots resulted in high adult mortality (69.8%) along with deformities in maggot and pupal stages (Fig. 3). Thus, it appears that rpl19-dsRNA is more effective to trigger RNAi silencing at adult stage of fruit fly. In contrast, despite to provide continuous supply of bacteria expressing dsRNA of either ECR or RPL19 gene for feeding to Bactrocera maggots did not result in overexpression of these target genes by formation of RNAi refractoriness as observed by Li et al. (2011) rather produced higher gene knock down affecting growth and development of Bactrocera adversely to survive very less fruit flies (13.1%) at last.
Gene silencing in terms of reduced relative transcript level of endogenous BdRPL19 gene was also observed after 15 days of feeding bacteria (700 μl) expressing dsRNA-RPL19 in treated 3rd instar maggots and subsequent emerged adult fruit flies which was reduced by 62 and 84%, respectively, than the native RPL19 gene expression in B. dorsalis (Fig. 3c).
Retarded development and abnormal phenotypes in different developmental stages of fruit fly
The feeding of bacteria expressing dsRNA of ecr and rpl19 individually to maggots of B. dorsalis produced similar marked physiological deformities. In the present study, typical phenotypic abnormalities such as absence of wings, underdeveloped abdomen and absence of legs, absence of complete abdomen, adults with severely curled wings and/or shrinked abdomen, malformed legs, and incomplete eclosion were observed in emerged adult B. dorsalis upon feeding E. coli expressing ecr-dsRNA or rpl19-dsRNA. It is very fascinating that similar types of phenotypic malformations have been observed earlier by treatment of Bactrocera with insecticides belonging to different chemical groups like Beticol, Biosad, Elsan, Lufox, Mani, Match, and Radiant. The tested insecticides reduced the fecundity and hatchability percentage and also caused high levels of sterility for adult females emerged from treated pupae (Halawa et al. 2013). Earlier, precocious lethal molt and several growth inhibitory effects were also observed by ingestion of tebufenozide by white tussock moth larvae and spruce budworm larvae (Dhandialla et al. 2005). Further, ECR is the target of bisacylhydrazine insecticides used against several pests (Billas and Moras 2005). The finding indicates the feasibility of orally delivered dsRNA method and silencing of these 2 potential genes of B. dorsalis to produce a proof of concept of RNAi gene silencing in fruit fly in laboratory conditions.
The maggot development, especially in dsRNA-ecr, was significantly affected and maggots produced were having change in melanization (gradual blackening on whole surface) and were reduced in size in comparison to control, and died prematurely. The maggots which survived were developed into puparium but produced change in melanization (black colored puparium) in contrast to normal pupae which are generally of tan or dark brown colored (Fig. 4a). Later on, the pupae which survived were converted into adult fruit flies but they showed shrunk appearance and underdevelopment, i.e., having underdeveloped body parts such as absence of wings and malformed legs, severely curled wings, underdeveloped abdomen and absence of legs, and in some cases complete abdomen was absent in comparison with control adult fruit flies which were normal in appearance (Fig. 4b). In case of control, normal maggot to pupa and pupa to adult developments were observed.
Mechanism of dsRNA-based gene silencing in B. dorsalis
A very high total mortality rate (including deformity) was observed in all the developmental stages of B. dorsalis, which has not been reported so far. It is well proven that plant incorporated dsRNA corresponding to an insect pest gene will have targeted activity against a particular insect pest as reported in western corn root worm (Bachman et al. 2016). The total average mortality (including deformity) in maggots, pupae, and adult B. dorsalis was little higher (87.9%) in maximum concentration (700 μl) of Bdrpl19-dsRNA bacterial treatment as compared than 86.3% in Bdecr-dsRNA bacterial treatment during the 2 consecutive years due to “loss of RNAi functions” phenotype (Saleh et al. 2006) and that ultimately resulted in death of flies. Based on earlier and present studies, it proves that oral administration of engineered HT115 bacterial strain is very much efficient to inhibit the target gene function in non-cell autonomous RNAi mode by transferring dsRNA without its degradation in insect body (Huvenne and Smagghe 2010). In the present study, RNAi effects of feeding bacteria expressing dsRNA within 15 days of start of feeding experiment was comparable as reported earlier by Li et al. (2011). But different typical phenotypic deformities observed in pupae and adult stages of B. dorsalis in our laboratory were not reported so far which validates the feasibility of this dsRNA expressing bacteria feeding method as more natural way of transferring intact dsRNA into insect body. As far as the roles of ECR and RPL19 genes are concerned, it was very obvious that RNAi effects produced due to silencing of these genes in whole body of B. dorsalis while not in particular tissues, despite to the uptake of corresponding dsRNA happens to be in midgut (Christiaens et al. 2020). In fact, different RNAi mechanisms of spreading the RNAi effector molecules could be possible in dipteran species; it can hypothesized that either dsRNA molecules along with artificial diet could be absorbed through the columnar cells having microvilli providing a large absorptive area with many channels and receptor-mediated endocytosis pathways (Joga et al. 2016) and after processing into siRNA molecules which actually moves to all other tissues and cells of B. dorsalis producing the silencing effects which ultimately resulted in phenotypic deformities in B. dorsalis. It was also very interesting that the sequence-specific dsRNA mediated silencing effects in terms of typical phenotypes were there from one developmental stage to another (Fig. 4).
Although, gene silencing effects were earlier reported in few studies, where feeding of bacteria expressing dsRNA to B. dorsalis adult targeting spermatogenesis reduced the number and length of spermatozoa stored in female spermathecal and significantly reduced the offspring hatching rate which further resulted in lesser number of maggots raised in fruits compared to control treatment (Dong et al. 2016). Similarly, feeding of dsRNA to B. dorsalis adults through artificial diet targeting spr resulted in 52% silencing in fruit flies and the mean life span reduced to 31 days in treated fruit flies (Zheng et al. 2015). Feeding of bacterially expressed dsRNA of four genes involved in ecdysteroidogenesis, i.e., ptth, torso, spook, and nm-g, showed knock down of these genes in Chilo suppressalis resulted in abnormal phenotypes, retarded development, and high larval mortality compared to control (Zhu et al. 2016). Thus, oral ingestion of bacterially expressed dsRNA into the body of insect seems to be a simple and appropriate mode to investigate potential management scheme for several important pests like B. dorsalis as observed in our study also.
In contrast to recently reported strong RNAi phenotypes in B. tryoni by complexing dsRNA targeting melanin synthesis gene, yellow with liposomes within the adult insect’s diet (Tayler et al. 2019), we hereby, report high gene-silencing effects in terms of strong RNAi phenotypes including typical deformities and very high mortality rates at maggot, pupae, and adult developmental stages of B. dorsalis just by ingestion of bacteria expressing dsRNA. This could be due to absence of either dsRNases in the gut or the transfer of intact dsRNA through feeding of E. coli expressing dsRNA of two non-midgut target genes in B. dorsalis. After the lysis of engineered bacteria inside the gut, these dsRNA could have processed into siRNAs, which is capable enough to target complementary mRNA transcripts of ECR or RPL19 gene present in different parts of body of B. dorsalis far away from the site of ingestion. This is sure that mechanism exists for the spread of RNAi signals in B. dorsalis as reported earlier. In fruit fly, systemic RNAi and dsRNA update is taken place through an active receptor-mediated endocytosis (Saleh et al. 2006). DsRNA is taken from the environment and then silencing signals are transferred through vesicle-mediated intracellular trafficking (Saleh et al. 2006; Tomoyasu et al. 2008). The dsRNA uptake by epithelial cells of the midgut in insects is very much significant for RNAi response. After midgut cells absorb the dsRNA, the transfer of these signals to intracellular RNAi machinery is also of paramount importance. The dsRNA uptake follows endocytosis pathway thus, enables its transfer to the cytoplasm through the discharge from endosome (Varkouhi et al. 2011).