Molecular changes accompanying the loss in fertility of Spodoptera frugiperda female moths following infection with sub-lethal dose of baculovirus

doi:10.21203/rs.3.rs-2308218/v1

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Molecular changes accompanying the loss in fertility of Spodoptera frugiperda female moths following infection with sub-lethal dose of baculovirus

https://doi.org/10.21203/rs.3.rs-2308218/v1

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Sub-lethal baculoviral infections are associated with several developmental and reproductive defects but the mechanism behind these defects is not well understood. Here, we investigated the reproductive defects induced by sub-lethal infection of Spodoptera frugiperda nucleopolyhedrovirus (SfNPV) in Spodoptera frugiperda (J.E.Smith) adults. We observed significantly less oviposition, loss of fecundity and egg hatchability in surviving adults after SfNPV infection. Reciprocal crosses with both the genders suggested that the infection of either sex could result in the fertility defects. An investigation of anatomy of reproductive of the surviving females suggested minor defects but confirmed reduced mating with infected males. Oviposition defects were observed in the infected group in spite of successful mating. Differential transcriptome analysis of infertile females from sub-lethally infected group revealed alteration of reproductive, developmental and behavioural pathway genes in comparison to control. We have also investigated the involvement of hormonal genes and piRNAs in their contribution to reproductive defects post sub-lethal baculoviral infection. This study will shed some light on the molecular mechanisms behind the sub-lethal effects observed in the surviving adults post baculovirus infection and will be useful to understand the infertility pathways in insects.

Fall armyworm (FAW), Spodoptera frugiperda (J.E.Smith) is a polyphagous Lepidopteran pest which has assumed the status of a global pest owing to its recent invasions in several countries including India (Nagoshi et al., 2019). Amongst the several biocontrol agents used for the management of FAW in India, Spodoptera frugiperda nucleopolyhedrovirus belonging to the family Baculoviridae was isolated from India in 2020 and was characterized for its insecticidal efficacy (Sivakumar et al., 2020). Baculoviruses are potent pathogens of several insect pests especially those belonging to the order Lepidoptera and are commercially used as bio-insecticides against major crop pests. Viral infection is initiated by the ingestion of polyhedral occlusion bodies (OBs) by insect larvae and typically culminate in insect mortality within 7–14 days of ingestion. The dead larvae are a source of amplified OBs and are required for horizontal transmission of the virus. Numerous studies have suggested that the larvae which survive the baculoviral infection can carry the infection sub-lethally in pupal and adult stages and can transmit it vertically to the next generation (Cabodevilla et al., 2011; Khurad et al., 2004). Thus, baculoviruses are known to adopt a mixed mode transmission where transmission to host is through vertical or horizontal route. (Williams, 2017). Sub-lethal baculovirus infections are not as comprehensively investigated as the lethal ones, but several studies associate them with slower developmental rates, reduced larval and pupal weights, altered sex ratios, decrease in fertility and fecundity in surviving populations (Perelle and Harper, 1984; Patil, 1989; Sait et al., 1994; Fuhr et al., 2021). Low to moderate levels of mortality have also been reported in the F1 generation of infected adults (Goulson & Gory, 1995; Monobrullah & Shankar, 2008; Patil, 1989). Thus, sub-lethal baculoviral infections can drastically reduce the reproductive potential of the surviving host and have a significant impact on population dynamics of the infected insect.

Despite the observance of gravid effects in multiple baculoviral host systems, very few studies have looked into the mechanisms leading to the debilitating effects of the virus. Most studies have primarily studied the effect of different viral concentrations and larval stages on the outcome and extent of sub-lethal effects (Milks et al., 1998; Monobrullah & Shankar, 2008; Fuhr et al., 2021). Delayed larval development and failure to molt is attributed to the hormonal changes induced by baculoviral infection. Juvenile hormone titers in infected Spodoptera litura larvae were found to be higher during late larval stages and were believed to be responsible for failure of larval to pupal molt (Subrahmanyam & Ramakrishnan, 1980). Baculoviruses are known to encode viral ecdysone UDP-glycosyl transferase gene (egt) which catalyzes the conjugation of sugar molecules to ecdysteroid hormone and renders it inactive, thereby preventing larval molting and pupation (O’Reilly & Miller, 1989). Lymantria dispar NPV infected larvae were found with higher hemolymph ecdysteroid levels which was found to interfere with larval development (Park et al., 1996). The studies looking into the changes at the pupal and adult level are even scarce. Sait et al. (1998) reported a decline in eupyrene and apyrene sperm numbers in granulovirus infected Plodia interpunctella males, however, the decline was not found to be dose dependent as opposed to the defects in male fertility (Sait et al., 1998). Helicoverpa zea adult females infected with a non-occluded baculovirus were found with severe reproductive defects and exhibited complete atrophy of the reproductive tract (Hamm et al., 1996). In this study, we have studied the reproductive defects in surviving adults post SfNPV infection and have tried to investigate the mechanisms leading to these defects. We have reported the transcriptomic changes in surviving adult females which failed to oviposit post infection. We believe this study might unravel the underlying causes behind sub-lethal effects of baculoviruses in surviving adults and might be a useful resource to understand the infertility mechanisms in insects.

2.1 Insect specimen and virus

Spodoptera frugiperda isolate used in this study was from ICAR-National Bureau of Insect Resources, Bengaluru and was isolated from Chikballapur, Karnataka, India. Spodoptera frugiperda nucleopolyhedrovirus strain was collected in September 2020 at a field near to Chikkaballapura, Bengaluru. India. The identity of SfNPV was established by amplification with polh specific primers. The nucleotide sequence was submitted to GenBank and the accession number was received (MT422725) (Sivakumar et al 2020). We used this viral inoculum for the entire study.

2.2 Diet surface contamination bioassay

Late 2nd instar larvae were used in this experiment and reared in individual plastic vials filled with 0.5 ml of artificial diet. Viral concentration was determined by using a Neubauer hemocytometer dark field at ×400 magnification. The diet was contaminated with 100µl of SfNPV viral suspension (10⁸ OBs/ml) by using a micro applicator and was allowed to air dry. Larvae were allowed to feed individually on the treated diets and only those larvae which had consumed the treated diet were included in the test and transferred to uncontaminated diet and reared up to adult stage. Death due to polyhedrosis infection were recorded at larval and pupal stages and the dead larvae/pupae were monitored for presence of occlusion bodies.

2.3 Assessment of reproductive potential

Pupa of control and SfNPV infected group were sexed and kept in separate containers. Virgin and freshly emerged adults (< 36hr old) were allowed to mate with the opposite sex within each group. For cross mating studies between infected and control groups, freshly emerged males/females of infected group were paired individually with the opposite sex of the control group. Adult males or females from infected group which showed defects in wing/abdomen development were excluded from the mating studies. Each pair was reared individually in a plastic container lined with white paper and covered with a muslin cloth. Adults were provided with a 10% honey solution which was renewed every 2 days. Observations were recorded every 24h on oviposition, number of eggs laid and number of eggs hatched.

2.4 Morphology of the reproductive tract and mating status

Adult females which failed to lay eggs were collected on Day6 of emergence. Adult females with wing malformation and without any wing malformation were selected to dissect and observe the morphology of their reproductive systems. The reproductive system was dissected and fat bodies were cleaned in phosphate buffer saline (PBS, pH 7.4). The bursa copulatrix, ovaries, spermatheca and spermathecal gland were examined for possible defects or distortions. The mated/non-mated condition of the adult females were confirmed by the observation of spermatophore inside the bursa copulatrix. Images were acquired by a Leica DMC4500 digital camera attached to a Leica M205A stereomicroscope.

2.5 Transcriptome analysis of control and infected adult female

Total RNA was extracted using TRIzol reagent (Invitrogen) in three biological replicates from a single female moth from two groups: (i) Control Day6 old fertile female and (ii) Day6 old female from infected group which had failed to oviposit. Genomic DNA contamination was removed using DNaseI digestion (Invitrogen). RNA quality assessment was done using RNA ScreenTape System (Agilent) in a 4150 TapeStation System (Agilent). 250ng of DNaseI treated RNA was used to enrich the mRNA using NEBNext Poly (A) mRNA magnetic isolation module (New England Biolabs) by following the manufacturers’ protocol. The enriched mRNAs were further taken for the library preparation using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (New England Biolabs). cDNA was amplified by 12 cycles of PCR with the addition of NEBNext Ultra II Q5 master mix, and NEBNext Multiplex Oligos for Illumina to facilitate multiplexing while sequencing. The amplified products were then purified using 0.9X AMPure XP beads (Beckman Coulter) and the final DNA library was eluted in 15µl of 0.1X TE buffer. The library concentration was determined in a Qubit.3 Fluorometer using The Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). The library quality assessment was done using Agilent D1000 ScreenTape System (Agilent) in a 4150 TapeStation System (Agilent). The library qualification criteria were based on the presence of a broad peak in the range of 300bp to 1000bp, with an average size of 300bp-450bp in the Agilent 4150 TapeStation system and the Qubit concentrations above 2ng/µl or 10nmol. The sequence data was generated using Illumina Novaseq. Data quality was checked using FastQC [Andrews S. (2010) FastQC: a quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/ fastqc) and MultiQC software (Philips et al., 2016). The data was checked for base call quality distribution, % bases above Q20, Q30, %GC, and sequencing adapter contamination.

Further, Raw data were processed to obtain high- quality reads, we used the open-source software package Trimmomatic (Bolger et al., 2014) to identify and to trim nucleotides falling below the established quality threshold (minimum 20 phred score) as well as to trim adapter sequences. The transcriptome de novo assembly was carried out using the Trinity program (Haas et al., 2013). The assembly was further refined using the tr2aacds pipeline in EvidentialGene package (http://arthropods.eugenes.org/EvidentialGene) which reduced the redundancy by selecting the assembled transcripts based on their coding potential leading to final non-redundant transcriptome assembly containing a unique set of transcripts. To check the quality of assembled transcriptome we analysed the coding capacity of assembly using BUSCO v 3.0.2 (Waterhouse et al., 2018). Further, Potential protein coding sequences were analyzed using Transdecoder (https://github.com/TransDecoder) to retrieve potential protein-coding regions.

For differential expression analysis, relative abundance of assembled transcripts was calculated using Kallisto v0.44.0 (Bray et al. 2016) which uses pseudo-alignment method to map transcript reads to targets for transcript abundance quantification and gives transcript estimates in transcripts per million (TPM). Further, DESeq program of R package (3.2.0) was used to measure differential expression for each transcript for fertile female and control samples. Genes with P- value 0.05 and log2-ratio ≥ 1.5 and P0.05 and log2- ratio≤ -1.5 were used as thresholds to evaluate the significant up and down-regulation respectively in the gene expression between control and infected females. To annotate the differently expressed genes, Blastx was performed against the NR database. The Gene Ontology enrichment analysis of DEGs was done using GOSeq software which corrects the gene length biases. The GO terms with over represented pvalue 0.05 were considered as significantly enriched. We used KOBAS 3.0 software to check the statistically significant enrichment of differential expression genes in KEGG pathways.

2.6 qPCR validation of mRNA transcripts and piRNA genes

Total RNA was extracted in five biological replicates from whole body of 6-day old fertile adult female of control group and 6-day old adult female of infected group which had failed to oviposit as described earlier. Besides this, total RNA was also extracted from three biological replicates of 6-day old females of infected group which had oviposited but showed defects in fecundity and egg hatchability. RNA concentration and quality was assessed using nanodrop spectrophotometer and agarose gel electrophoresis. First strand cDNA was synthesized using 1µg total RNA and PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan) according to kit manual. qPCR was performed with atleast three biological replicates of each sample using TB Green Premix Ex Taq II kit (TaKaRa, Shiga, Japan) in three technical replicates. 28S rRNA gene was used as an endogenous control. The list of gene specific primers used in this study are provided in Additional File 1. For qPCR of piRNA genes, stem-loop RT primers were designed for each piRNA gene and for bmo-miR-2766-3p, Sfr-miR-14-5p and Sfr-miR-184-3p. bmo-miR-2766-3p was used as an endogenous control in this study. Briefly, 5pmol of piRNA RT primer and 5 pmol of bmo-miR-2766-3p RT primer were added instead of RT primer mix during first strand cDNA synthesis in each sample. The remaining procedure was followed according to Primescript RT Reagent kit with gDNA eraser manual (TaKaRa, Shiga, Japan). qPCR was performed using Universal Reverse primer and small RNA specific forward primer (Additional File 1) using TB Green Premix Ex Taq II kit (TaKaRa, Shiga, Japan). qPCR was performed on CFX-96 Real time PCRdetection system (Bio-Rad) and was followed by a melt curve analysis. Data analysis was carried out with Bio-Rad CFX maestro software (ver. 4.1 BioRad, Hercules, CA, USA). Relative expression levels of the genes of interest were normalized relative to the abundance of the reference gene using the ΔΔCq method (Livak & Schmittgen, 2001).

2.7 Statistical analysis

The fecundity and egg hatchability data from control and infected group was subjected to normality test distribution using Shapiro-wilk test. Since fecundity data had passed the normality test, it was subjected to Welch’s t test to test the significance between control and infected groups. Egg hatchability data did not meet the assumptions of normality distribution and was subjected to Mann-Whitney test to estimate the p-value. Oviposition data was subjected to Chi-square test to check the association of non-oviposition with the infected group. Reciprocal crosses between males and females from control and infected groups were subjected to Dunnett’s T3 multiple comparisons test to test the significance. Hatchability data of reciprocal crosses was tested using Kruskal-Wallis test. The statistical analysis and data visualization was done using GraphPad Prism 9.4.1. qPCR data was analysed and tested for statistical significance using CFX maestro software (ver. 4.1 BioRad, Hercules, CA, USA).

3.1 Reproductive potential of the adults surviving SfNPV infection

We were interested to examine the reproductive potential of insects surviving sub-lethal doses of baculoviral infection. For this, we infected the late second instar larvae of Spodoptera frugiperda with 10⁷ occlusion bodies (OBs) of Spodoptera frugiperda nucleopolyhedrovirus using diet surface contamination method. This dose was previously determined to lead to sub-lethal infections with significant reproductive defects at adult stages (Manuscript submitted). The larvae infected at the said dose exhibited typical symptoms of baculoviral infection including slower developmental rate, liquification followed by death in 7–14 days. At the administered dose, we observed nearly 25% larval mortality, whilst 37% mortality was observed at the pre-pupal and pupal level (n = 177). The dead larvae were examined for SfNPV infection by observing for the presence of occlusion bodies. The sub-lethally infected larvae were also checked for the presence of occlusion bodies in hemolymph. All tested larvae (n = 20) demonstrated presence of occlusion bodies in hemolymph while the control group did not show the presence of occlusion bodies and the associated mortality. The surviving larvae were sexed at pupal stages and kept for emergence of adults in separate containers. We observed a large percentage of adults (25%, n = 79) exhibiting structural wing deformities in sub-lethally infected group when compared with control group. One day old male and female adult moths without structural defects were paired in control and infected groups respectively to study their reproductive potential. Each pair from the respective group was prospected for several reproductive traits including their ability to lay eggs, fecundity (number of eggs laid/female) and percentage hatchability of eggs. We found significant differences across the two groups in the defined parameters. In the control group, all females laid an average of 450 eggs by Day4 and Day5 of emergence (n = 22). While in the infected group, 58.7% of the females were unable to lay eggs even till Day6 of emergence (n = 63) (p < 0.0001) [Figure 1B]. By observation, the infected females which did not lay eggs by Day6 were subsequently found to be infertile in their lifespan. Further, considerable difference was found between fecundity of the two groups with infected group females demonstrating 60% lower fecundity than the control group [t(40.72) = 9.4; p < 0.0001]. The hatching percentage of the eggs in the two groups was also significantly different. Median hatching percentage of infected group of females was 40% lower than the control group [Mann Whitney U = 3, n1 = 20, n2 = 24, p < 0.0001 two tailed). A number of egg masses (n = 8/24) in the infected female category were sterile and did not give rise to any offsprings while all egg masses from control group were found to be fertile. In all, we observed drastic reduction in the reproductive potential of the sub-lethally infected group with respect to the control and were interested to examine the potential reasons behind the observed effect.

3.2 Cross-mating between infected and control adults:

Next, we tried to examine the cross-mating between the different sexes of infected adult moths and control moths to understand the productivity of these crosses. We paired the adult moths in four groups: (i) Control female x Control male (ii) Infected female x Infected male (iii) Infected female x Control male (iv) Infected male x Control female. Barring the cross between control male and control female, all other crosses resulted in infertile pairings, significantly reduced fecundity and egg hatchability (Fig. 2). This suggests that baculovirus infection of either male or a female moth is sufficient to impact the fertility and reproductive outcome of the cross.

3.3 Evaluation of mating status and anatomy of reproductive system of SfNPV infected adult females

The female reproductive system of control Spodoptera frugiperda was observed to have a pair of ovaries (each with 4 ovarioles), oviduct, spermatheca, spermathecal gland and female accessory glands as shown in Fig. 3A. We observed the reproductive tract of a number of females from control group on Day6 of emergence (n = 10) and found that all the analysed females possessed a well-developed reproductive system. We checked whether the females had mated during their pairing by looking for the presence of spermatophore in their Bursa copulatrix (Fig. 3B). All females of the control group (n = 10) were found to have a spermatophore in their Bursa copulatrix. Next, we analysed the females of infected group on Day6 of emergence. As described earlier, a number of females in the infected group category exhibited structural deformities in their wing formation and were not used for mating studies. When we observed the reproductive tract of these females, we found that all of the adult females with wing malformation had severe distortion of their reproductive system. The ovaries were not intact and the entire reproductive system appeared to be heavily collapsed (n = 9) (Fig. 3D). We then analysed the reproductive tract of the infected group females which had not laid eggs by Day6 of emergence and which did not show any wing malformations. Although a majority of the females possessed an intact reproductive tract (n = 9/11), some of the adult females from infected group (n = 2/11) were found to exhibit atrophy with distortion of ovaries, however all other parts of the reproductive system like bursa copulatrix, spermatheca, spermathecal gland were observed to be present (Fig. 3C). We were particularly interested to know whether the infected females had mated during their pairing since these had not laid any eggs by Day6. Out of 11 tested females, we observed that five females had spermatophore in their Bursa copulatrix indicating successful mating with infected male (Fig. 3C), while six females showed absence of mating. It was interesting to observe that even after successful mating, the female adults failed to lay eggs. Our results suggest that baculovirus infection can affect the reproductive ability of their hosts either by affecting their ability to mate or through post mating mechanisms.

3.4 Transcriptome analysis of control and infected adult females

In order to understand the molecular changes accompanying the loss in fertility in baculovirus infected adult females, we performed transcriptome sequencing of fertile control females and infected females which failed to lay eggs by Day6 of emergence in three biological replicates each. We generated a total of 47.2GB data from the six libraries with %Q30 ranging from 96.1–98.27%. After removal of adapter reads and low-quality data, we checked for differential gene expression profiling using DEseq2 and observed upregulation of 190 genes and downregulation of 97 genes in the infected female transcriptome with Log₂Fold change > 1.5 and p-value < 0.05 (Additional File 2). Using ncbi nr database, we could annotate 180 differentially expressed genes within our data. Although most of the annotated genes did not possess known functions, we could fetch a number of differentially expressed genes with known involvement in reproductive pathways. Some examples of the genes upregulated in infected females included UDP-glucoronosyltransferase 1–2 like, protein doublesex-like, acyl CoA Delta(11) desaturase-like (XP_035433515.1), protein gooseberry-neuro-like, putative endonuclease/reverse transcriptase, REPTOR-binding partner among others. Some examples of the downregulated genes included CAPA peptides like, chitin synthase chs-2 like, cytochrome P450 307a1-like, protein yellow like, ejaculatory bulb-specific protein 3-like, transcription factor Adf-1 like, acyl CoA Delta(11) desaturase-like (XP_035433488.1) etc. We performed Gene Ontology and KEGG pathway enrichment analysis of differentially expressed genes to understand the mechanisms leading to infertility in infected females. Amongst the significantly enriched biological pathways in GO analysis, biological regulation, developmental process, reproductive process, immune system and behaviour were highly represented (Fig. 4A). We found RNA transport, Ubiquitin mediated proteolysis, apoptosis, nucleotide excision repair, Dorso-ventral axis formation, neuroactive ligand-receptor interactions amongst the significantly enriched KEGG pathways in our data (Fig. 4B). The results indicated that several reproductive, developmental and behavioural pathways were manipulated in surviving adults post baculoviral infection.

We tested the expression of UDP-glucoronosyltransferase 1–2 like (UGT), REPTOR-binding protein, acyl coA Delta (11) desaturase-like (XP_035433515.1) (D11), ejaculatory bulb-specific protein 3- like (ebulb), protein doublesex like protein (dblsex), uncharacterized protein KIAA0930 homolog (XP_022829090.1) (930), octopamine receptor beta-2R (octb2r) with qPCR. With the exception of protein doublesex like protein, rest of the genes followed a similar trend of regulation as predicted by RNAseq analysis. However, for most of the genes tested in qPCR, we could achieve borderline significant or non-significant trends. Uncharacterized protein KIAA0930 homolog (p = 0.08), protein doublesex like (p = 0.04), octopamine receptor beta-2R (p = 0.09), REPTOR-binding protein (p = 0.05), ejaculatory bulb-specific protein 3- like (p = 0.1) were amongst the genes with notable trends in qPCR analysis (Fig. 4C).

We next compared the expression of the validated genes in sub-lethally infected female moths which had oviposited and which failed to oviposit. Though both the category of infected female moths were found with reproductive defects, the nature of defects was different. Former exhibited defects in fecundity and egg hatchability while the latter did not lay eggs at all. REPTOR-binding protein like gene was found to be significantly upregulated (p = 0.02) in females which had failed to oviposit. The other genes did not display statistically significant results in our analysis (Fig. 4D).

3.5 Regulation of hormonal genes in infected females with reproductive defects

Since baculoviral infection is known to interfere with insect hormonal machinery (O’Reilly & Miller, 1989), we were interested to study the regulation of hormonal genes in infected adult females. Since we did not find hormone related genes in transcriptome data with our threshold limits, we decided to check the regulation of insect hormone related genes directly through qPCR. We tested the regulation of ecdysone receptor, ecdysone inducible protein E75, nuclear hormone receptor HR3, juvenile hormone binding protein, juvenile hormone esterase gene in control and infected females. As observed with previously tested genes, we did not observe statistically significant results for the tested hormonal genes. Nuclear hormone receptor HR3 was upregulated by 2.6 folds in infected females which had failed to lay eggs (p = 0.18). Ecdysone inducible gene E75 was downregulated by 0.75 folds (p = 0.08).

When we tested the expression of these genes within infected females with and without oviposition defects, we observed that several insect hormone related genes were significantly dysregulated in the two groups. Nuclear hormone receptor HR3 was 10-fold upregulated (p = 0.01) in sub-lethally infected females which had failed to lay eggs. Ecdysone inducible gene E75 and ecdysone receptor gene were upregulated by 2.2-folds (p = 0.03) and 5.62-folds (p = 0.03) in infected group with oviposition defects. Juvenile hormone esterase gene was 5.5-fold upregulated in infected female group with fecundity and egg hatching defects (p = 0.1). Overall, hormonal genes between the two categories of infected females showed marked differences in their expression pattern.

3.6 Dysregulation of piRNAs in infected females with reproductive defects

In previous studies, infection of insect cells with baculoviruses has been associated with profound downregulation of piRNAs and upregulation of transposon expression (Kharbanda et al. 2015, Nayyar et al., 2017). We observed upregulation of reverse transcriptase and retroelement polyprotein in transcriptome of infected females (Additional File 2). Since piRNA dysregulation is known to contribute to infertility and reproductive defects, we were interested to study the expression of piRNA in infertile infected females in our study. We selected some piRNAs that were found to be downregulated in small RNA libraries of baculovirus infected Sf21 cells (Kharbanda et al. 2015) and studied their expression in fertile control and infertile infected adult females using stem-loop RT-PCR. We found that all the tested piRNAs were markedly downregulated in infertile infected females. piR-71087 showed the highest downregulation (nearly 8-fold, p < 0.05) amongst the tested piRNAs while piR-43773 was 6-fold downregulated in infected females (p = 0.009) (Figure). bmo-miR-2766-3p was used as an endogenous control while calculating the relative expression (Kharbanda et al., 2015). To verify the specific downregulation of piRNAs in the infected samples, we designed stem loop RT-PCR primer for two endogenous host miRNAs, Sfr-miR-14-5p and dme-miR-184-3p and tested their expression in control and infected adult females. Both the miRNAs did not show any trend of downregulation in infected females.

Further, we checked the expression of the tested piRNA genes in sub-lethally infected female group with oviposition defects and fecundity defects respectively. We observed that females showing fecundity and egg hatchability defects had similar to lower level of piRNA gene expression than the infected female group showing failure in egg laying. piR-33468 and piR-35413 were upregulated by 4.23-folds (p = 0.17) and 4.66-folds (p-0.15) respectively in the infected females which had failed to lay eggs.

We had previously studied the sub-lethal effects of different concentrations of Indian isolate of SfNPV on larval and pupal development, reproductive potential, adult lifespan and vertical transmission in S. frugiperda (Manuscript submitted). In this study, we have extensively characterised the effect of SfNPV on reproductive potential of the host and have tried to investigate the basis for reproductive defects observed in adult female insects following infection with sub-lethal dosage of SfNPV. To begin with, we examined the extent of reproductive damage inflicted by NPV infection in S. frugiperda. We observed increased incidences of infertile pairings between crosses of infected adults along with significant reduction in fecundity and egg hatchability when compared to the uninfected adults. The magnitude and nature of defects observed were in line with the reproductive defects reported in several other insects following baculovirus infection (Cabodevilla et al., 2011; Fuhr et al., 2021; Patil, 1989; Rothman and Myers, 1994). Next, we wanted to examine the role of gender in reproductive outcomes following infection. We observed impaired reproductive outcomes when either male or female adult was infected. An earlier study had also reported reduced egg viability in S. littoralis when NPV infected males or females were paired with the uninfected ones although no effect was found on fecundity in either case (Vargas-Osuna and Santiago-Alvarez, 1988). Sait et al. (1998) reported a dose dependent decrease in fertility of males after sub-lethal infection with Plodia interpunctella virus when crossed with uninfected females. Cross mating of Bombyx mori NPV infected females with uninfected males resulted in significant reduction in fecundity and hatching of eggs (Khurad et al., 2004). Thus, our results are in line with other previous studies which indicate that both sexes show reduced reproductive ability after sub-lethal infection with baculoviruses.

Further, since both the genders were impacted post baculovirus infection, in this study we focussed our attention to the changes in SfNPV infected females. We tried to examine the anatomy of the female reproductive system to observe any gross changes post infection. It was earlier observed that 62.8% of Helicoverpa zea adult females infected with a non-occluded baculovirus were agonadal at Day3 of oviposition with a waxy plug protruding from their vulva (Hamm et al., 1996). However, Spodoptera littoralis granulovirus was not found to alter egg production or morphology of female reproductive tract after infection (Hatem et al., 2011). In our study, we did not observe gross structural defects in anatomy of female reproductive tract in majority of the females from sub-lethal group, nor did we observe the presence of viral or mating plug. Further, we also observed that the baculovirus mediated fertility defects could arise because of altered mating behaviour of the infected adults or due to lack of oviposition after mating. Previously, similar to our study, Spodoptera littoralis granulovirus, infected adult pairs were found with reduced mating efficiency or inability to lay eggs in spite of mating (Vargas-Osuna and Santiago-Alvarez, 1988). Orcytes rhinoceros male adults were found to mate less often after baculovirus infection (Zelazny, 1977).

We performed differential gene expression profiling of fertile control female adults and SfNPV infected females which had failed to lay eggs by Day6 of emergence. Upon gene ontology analysis, we discovered significant enrichment of genes in several reproductive, developmental and behavioural pathways. Baculoviruses are well known to induce behavioural changes in infected larvae. They have been found to increase locomotory behaviour of infected larvae through the expression of viral protein tyrosine phosphatase gene (Kamita, 2005). They are known to upregulate the expression of odorant receptors, SexiOR35 and SexiOR23 in infected larvae, thereby altering the insect chemoreception (Llopis Gimenez et al., 2021). In our study, we found upregulation of acyl Delta(11) desaturase gene in infected adults which is known to mediate formation of delta₁₁ fatty acyl precursors for pheromone biosynthesis in pheromone gland (Tillman et al. 1999). Previously, Hz-2v virus infected females were found to release significantly higher pheromones than control females which was speculated to aid in viral transmission (Burand et al., 2005, Burand and Tan, 2006) Alternately, pheromone synthesis can also be higher in virgin females than the mated ones, and since some females in the infected category had not mated, it could account for higher levels of acyl D11 desaturase gene expression in infected females. But we noticed elevated levels of D11 desaturase gene expression in ovipositing infected females as well. Thus, we speculate that the baculoviral infection might either increase pheromone biosynthesis or might have an affect on post-mating rejection behaviour of the infected females, whereby pheromone levels were not reduced post mating. Another gene which is known to be critical for female sexual behaviour is doublesex gene and we observed significantly reduced expression of this gene in infected females in qPCR data. Disruption of synaptic activity of dsxGal4-expressing neurons was associated with complete infertility in females. These females actively rejected the male courtship responses and displayed aberrant copulation behaviour. In spite of the sperm transfer, these females did not lay eggs and were capable of remating indicating a suppression of postmating responses (Rideout et al., 2010; Rezaval, 2012). A number of these phenotypes were reminiscent of our observations and we speculate a role of dysregulated dsx gene in baculovirus mediated fertility defects in females. We also observed upregulation of octopamine receptor beta-2R gene in infected females in our study. Octopamine is a biogenic amine which plays a crucial role in several reproductive organs like ovaries, oviducts and sperm storage organs (Monastirioti, 2003; Rodriguez-Valentin et al., 2006). Octb2r homozygous mutant females were found to be sterile with reduced ovulation (Lim, 2014). Thus, potential role of octopaminergic neuromodulation in infected females could also be tested.

Several studies have reported modulation of insect hormonal levels during larval development after baculovirus infection. Ecdysteroid titer was found to be higher than controls in Lymantria dispar NPV infected larvae during fourth to fifth larval molt, but remained unchanged during larval to pupal molt (Park et al., 1993). The hemolymph juvenile hormone titer in NPV infected Spodoptera litura was found to be higher than the control titer and was suggested to result in its failure to pupate (Subrahmanyam and Ramakrishnan, 1980). Baculovirus infection of Heliothis virescens resulted in upregulation of nuclear hormone receptor HR3, juvenile hormone epoxide hydrolase and downregulation of juvenile hormone suppressible protein and juvenile hormone diol kinase in hemocytes (Nguyen et al. 2013). We also observed an upregulation of ecdysone pathway related genes (HR3, E75, EcR) in infected females showing fecundity defects. There was also a trend of hormonal dysregulation in control and infected females, however, the same was not statistically significant. Ecdysone signalling plays a critical role at different stages of oogenesis, ranging from proliferation and differentiation of germline stem cells, stem cell niche maintenance, ovarian follicle development, integration of nutrient status and environmental cues to govern egg fate at key checkpoints (Ables and Barbosa, 2010; Bownes & Blair, 1986; Gancz et al. 2011; Grunkteko et al., 2003) Ecdysone levels are believed to be higher in conditions of environmental and nutrient stress and are known to contribute to apoptosis of egg chambers (Rauschenbach et al., 2000; Terashima et al., 2005). Our results suggest dysregulated ecdysone levels in ovipositing infected females and warrants an investigation in its role in fertility defects post baculoviral infection.

Baculovirus infection of Sf21 cells was found to upregulate Transposase and PiggyBac transposable element proteins during early and late stage of infection (Nayyar et al., 2017). Several retroviral proteins/errantiviruses have also been found to be upregulated upon baculoviral infection of insect cells in multiple studies (Breitenbach, 2011; Nguyen, 2013). Our transcriptome data suggests upregulation of reverse transcriptase and retroelement proteins in infected female adults. Previous studies have also suggested downregulation of piRNAs upon baculovirus infection of insect cells (Kharbanda et al., 2015, Mehrabadi et al., 2013). Since piRNA dysregulation is known to contribute to infertility, we wanted to examine the importance of piRNA pathway in infertile phenotypes of baculovirus infected adult females Batista et al, 2008; Guan & Wang, 2021). We observed statistically significant downregulation of piRNAs in infected females showing fertility defects. From our data, it appears that piRNAs might have a role in infection mediated loss in fecundity/egg hatching possibly because of compromised genomic integrity of the developing embryos.

In this study, we have tried to understand the baculovirus mediated loss in fertility of surviving S. frugiperda females by examining their reproductive anatomy, mating potential and transcriptome changes with respect to control females. We suggest a role for behavioral and neuronal changes, hormonal and piRNA dysregulation as possible mechanisms affecting the female fertility post infection.

FAW

Fall Armyworm

SfNPV

Spodoptera frugiperda nucleopolyhedrovirus

occlusion bodies

UGT

UDP-glucoronosyltransferase 1–2 like

D11

acyl coA Delta (11) desaturase-like

ebulb

ejaculatory bulb-specific protein 3- like

dblsex

protein doublesex like protein.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets supporting the conclusions of this article are available in the NCBI SRA portal as BioProject ID: PRJNA890925.

Competing Interests

The authors declare that they have no competing interests.

Funding

The study has been funded by DST-INSPIRE Faculty Grant from Department of Science and Technology, India.

Author’s contributions

NN, AG, RRS, AA conducted the experiments and performed the analysis. NN drafted the manuscript. NN, TV, GS, MM and RG gave useful suggestions and helped in the overall plan of the study.

Acknowledgements

We would like to thank the Director ICAR-National Bureau of Agricultural Insect Resources, Bengaluru, Karnataka, India for the facilities provided for conducting the study.

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No competing interests reported.

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Molecular changes accompanying the loss in fertility of Spodoptera frugiperda female moths following infection with sub-lethal dose of baculovirus

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Abstract

Figures

1. Background

2. Methods

2.1 Insect specimen and virus

2.2 Diet surface contamination bioassay

2.3 Assessment of reproductive potential

2.4 Morphology of the reproductive tract and mating status

2.5 Transcriptome analysis of control and infected adult female

2.6 qPCR validation of mRNA transcripts and piRNA genes

2.7 Statistical analysis

3. Results

3.1 Reproductive potential of the adults surviving SfNPV infection

3.2 Cross-mating between infected and control adults:

3.3 Evaluation of mating status and anatomy of reproductive system of SfNPV infected adult females

3.4 Transcriptome analysis of control and infected adult females

3.5 Regulation of hormonal genes in infected females with reproductive defects

3.6 Dysregulation of piRNAs in infected females with reproductive defects

4. Discussion

5. Conclusion

Abbreviations

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