Analysis of maternal effect genes from maternal mRNA in eggs of Sogatella furcifera

To understand how many kinds of mRNAs in female adults can be transferred into the eggs and the molecular basis of embryonic axis specification in Sogatella furcifera, we performed de novo transcriptome sequencing of six cDNA libraries of female adults and unfertilized eggs. Total 60,306 unigenes were obtained, with an average length of 1114.51 bp and N50 length of 2112 bp. Total 2900 differentially expressed genes with 496 upregulated and 2404 downregulated were found in unfertilized egg compared to female adult. Most of mRNAs in female adult could be passed into its eggs. Total 65 maternal effect genes were identified, including many homologous genes involved in embryonic axis specialization of D. melanogaster. This study provide transcriptome resources to elucidate the functions of maternal effect genes and the molecular basis of embryonic axis specification in S. furcifera in the future.


Introduction
Insect maternal effect genes mean that these genes are expressed in the insect mother, and then the expression product (mRNA or protein) is transported into the egg, which plays a variety of roles such as regulating or affecting the early development, later growth, development process of the embryo, and adult phenotype [1].Early studies on fruit flies found that if the mother with the mutant gene mated with the father with the wild gene, the resulting offspring would still be mutant, thus confirming the existence of the maternal effect gene.Further studies have shown that maternal effect genes are very important for early embryonic development and can regulate the differentiation of anterior/posterior and dorso-ventral polarity [2].In recent years, with the rapid development of sequencing technology [3] and the improvement and popularization of RNAi technology [4], it has been found that maternal effect genes play an important regulatory role in the early development of embryos.It can also affect and regulate the frequency and degree of diapause occurrence, enhance the resistance to foreign invasion (especially microbial infection), and regulate the occurrence, development time, growth rate and survival status of the offspring [5].
The white-back planthopper is an important rice pest, belonging to Homoptera, Planthopper family.Although there are many researches about this pest, they mainly focus on the biological and ecological characteristics, dimorphism of wings, control methods, etc., and rarely involve the study of early embryonic development and maternal effect genes.It is known that maternal effect genes are very important for early embryonic development and can regulate the differentiation of anterior/posterior and dorso-ventral polarity [2], so the identification of maternal effector genes will be helpful to the understanding of embryonic development and axial differentiation of the white back planthopper.In this study, we attempt to sequence and analyze the maternal mRNA in the unfertilized eggs of white planthopper to determine how many kinds of mRNAs in female adults can be transferred to the eggs.At the same time, bioinformatics analysis was used to determine how many kinds of the maternal mRNAs are the product of genes that have been shown to be maternal effects in other species, and to infer the anterior/posterior pattern specification of early embryonic development compared with Drosophila.

Sequence assembly and functional annotation
Three biological replicates for female adult samples and their unfertilized eggs samples were designed for transcriptome sequencing.Total 123,160,466 clean reads were generated for six libraries.The average Q30 value of these libraries was 95.25 %, indicating the sequencing data were effective and reliable.The mean length and N50 length of total 60,306 unigenes was 1114.51bp and 2112 bp (Table 1).25,509 and 11,681 unigenes had homologous sequences in the Nr and Swiss-Prot protein databases, while 7,193,22,930,17,247,15,748,20,531, 28,172 and 18,592 unigenes could be classified by COG, GO, KEGG, KOG, Pfam, TrEMBL, and eggnog databases, respectively.Total 30,116 unigenes were annotated, of them 300 ≤ length<1000 and length ≥ 1000 unigenes were 10,110 and 15,770 (Table 2).There were 50,075 unignes in unfertilized eggs samples, just a little less than 54,586 unignes in female adult samples (Table 2).It suggest that most of mRNAs in female adult could be passed into its eggs.

Table 3
The list of top 10 up-regulated genes in unfertilized eggs than female adults.

Discussion
S. furcifera is an important agricultural pest in china and Southeast Asian countries.It cause grievous damage to many crops, such as rice, wheat, corn and sorghum, etc. [7].The serious threat to food production and security result from its robust fecundity and viruses transmitting, etc. [8].Maternal effect genes play an important regulatory role in the early development of embryos, and can regulate the occurrence, development time, growth rate and survival status of the offspring [5], so they have a lot to do with pest fecundity.Although many researches on S. furcifera have been done, the detail maternal effect genes and embryonic axis specification mechanisms of S. furcifera is still unclear.
In this study, a de novo transcriptome was assembled with sequences from female adults and unfertilized eggs.A total of 2900 DEGs were identified, among which 496 DEGs exhibited relatively higher expression levels in the unfertilized eggs than the female adults (Fig. 1).Total 65 maternal effect genes have been identified according to GO and Nr notification enrichment results, these genes should have some functions involved in one or more of anterior/posterior axis specification, dorsal/ventral axis specification, cell projection morphogenesis, lethal maternal effect, maternal effect embryo arrest, etc.
The unfertilized eggs were dissected from the female adults, so all mRNAs in the unfertilized eggs were from the female adults.Hence, the up-regulated expression DEGs mean that these genes were expressed in the female adults, then mainly passed and stored in eggs.The up-regulated expression DEGs included many enzymes, such as 3-isopropylmalate dehydratase, squalene synthase, carboxylesterase, pancreatic triacylglycerol lipase, etc. (Table 3).We know egg formation and early embryogenesis is dependent on stored molecules deposited by the mother.There are many kinds of enzymes in these stored molecules, and they have been verified to play an important role in early embryogenesis of some insects [9,10].The functions of up-regulated expression DEGs in early embryogenesis of S. furcifera are not clear and need to be investigated in the future.The down-regulated expression DEGs mean that these genes were expressed in the female adults, and less stored in eggs.DEGs of the down-regulated expression should have some role in adult development, but less in early embryogenesis.For example, longwave-opsin showed extremely high relative ratio in adult stages of Dendrolimus punctatus Walker to correlate to the nocturnal lifestyles of this species at adult stage [11]; odorant-binding protein 2 was primarily expressed in the antennae of Batocera horsfieldi (Hope) adults to play a key role in insect olfaction [12]; clavesins are expressed exclusively in neurons to provide a unique neuron-specific regulation of late endosome/lysosome morphology in Clavesin Family [13].
Insects have evolved many mechanisms for establishing embryo polarity that are based on maternal mRNA localizations at the anterior pole or both the anterior and posterior poles of the egg [14].Most of 65 maternal effect genes in S. furcifera have been found to be related to anterior/posterior axis specification or dorsal/ventral axis specification in terms of function in D. melanogaster, such as exuperantia, staufen, vasa, pumilio, tudo, dorsal, etc.It mean that the embryonic axis specification in S. furcifera somewhat similar with D. melanogaster.
There are mainly 10 genes involved in the posterior polar specialization of Drosophila posterior, including polenanos、tudor、 oskar、vasa、valois、pumilio、caudal、staufen、cappuccino、spire.In S. furcifera, 8 homologous genes except oskar and vasa were found (Table 5), suggesting that the two insects are very similar in terms of posterior polar specialization.There are mainly 3 genes involved in the anterior polar specialization of D. melanogaster, including bicoid、exuperantia、swallow.The anterior-to-posterior Bicoid gradient polarizes this process along the primary axis by driving chromatin accessibility at cis-regulatory elements of target genes that need to be activated for anterior specification [15].However, in S. furcifera, only exuperantia were found, not bicoid and swallow (Table 5), suggesting that there are some difference in terms of anterior polar specialization between S. furcifera and D. melanogaster.
In fact, among the lower dipterans (Nematocera), moth flies and culicine mosquitoes evolved new anterior determinants that are encoded by unrelated C2H2 zinc-finger genes, including zic/odd-paired and cucoid, respectively [2], not the anterior determinant gene bicoid from a duplicated Hox 3 ortholog in D. melanogaster [16].It is interesting that we also did not find zic/odd-paired and cucoid in

Table 4
The list of top 10 down-regulated genes in unfertilized eggs than female adults.S. furcifera, suggesting that the anterior specification of S. furcifera is also different with the lower dipterans (Nematocera), moth flies and culicine mosquitoes.Therefor the mechanism of S. furcifera anterior specification will need to be further explored in the future.

Conclusion
We performed de novo transcriptome sequencing of six cDNA libraries of female adults and unfertilized eggs of S. furcifera.We assembled total 60,306 unigenes and annotated them by searching for homology in protein databases.Most mRNAs in female adult could be passed into its eggs.Total 2900 differentially expressed genes (DEGs) with 496 upregulated and 2404 downregulated in unfertilized egg compared to female adult, and 65 maternal effect genes were identified, including many homologous genes involved in the anterior and posterior polar specialization of D. melanogaster.These transcriptome data provided a fundamental support for future functional studies to elucidate the functions of maternal effect genes and the molecular basis of embryonic axis specification in S. furcifera and other species.

Insect culture and samples collection
Successive generations of the white-backed planthopper strain were reared on rice seedlings.The culture condition was 28 ± 2 • C with a 16:8 h light: dark cycle.The 5-instar female nymphs with long-winged bud were selected and raised in a cage alone.When they emerged into adults and grew to the point of abdominal hypertrophy (when the eggs were mature), the adults were dissected to take out the mature eggs.The eggs were washed three times using physiological saline and collected as samples of unfertilized egg, and the remaining body parts were taken as samples of female adult.Three biological replicates were set up.Each group samples of female adult included seven individuals, and the egg numbers of the three groups of unfertilized egg samples were 231, 224, and 228, respectively.All samples were snap-frozen in liquid nitrogen and stored at − 80 • C prior to RNA extraction.

RNA isolation and sequencing
Total RNA of each sample was isolated using Trizol reagent according to manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).The concentration and purity of RNA was measured using Qubit® RNA Assay Kit in Qubit®2.0Flurometer (Life Technologies, CA, USA) and the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), respectively.The integrity of RNA was checked by electrophoresis in 1 % agarose gel and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).The RNA with 260/280 nm ratio between 2.0 and 2.1 was utilized to construct the cDNA library, then the qualified library was sequenced by the high-throughput sequencing platform with PE150 mode.

De novo transcriptome assembly and annotation
All the raw data has been submitted to NCBI Sequence Read Archive with accession numbers SRP474194 (female adult: SRR26948444, SRR26948443, SRR26948442; unfertilized egg: SRR26948441, SRR26948440, SRR26948439).The clean reads were obtain through removing the raw reads with adaptor contamination, low quality, and ambiguous base 'N' larger than 5 % by a custom Perl script.De novo assembly of short reads was accomplished by Trinity [6].Functional annotations were performed by the sequence comparison of unigenes with public databases included NR, Swiss-Prot, TrEMBL, COG, KOG, eggNOG 4.5, Pfam, GO, and KEGG using the BLAST algorithm with a cutoff E value of <10 − 5 , respectively.

Identification of differential expression genes (DEGs)
Differential expression analysis of female adult samples and unfertilized egg samples was performed using differential analysis software DESeq2.Criteria for differentially expressed genes was set as Fold Change (FC) ≥2 and False Discovery Rate (FDR) < 0.01.FC refers to the ratio of gene expression in two samples.FDR refers to the adjusted p-value used to measure the significance of the difference.In order to facilitate comparison, take the logarithmic value of the fold change as log2FC.

Identification of maternal effect genes
In order to authenticate genes concerned with maternal effect in S. furcifera, we firstly found out these genes from the GO enrichment result of all unigenes and referred to the annotation results from NR blasted with similar maternal-effect genes already reported.

Fig. 1 .
Fig. 1.Volcano plot of DEGs between the unfertilized eggs and female adults samples.The x-axis indicates log2 FC between the two samples and the y-axis indicates the -log10 (FDR) of gene expression variation.Both red and blue dots show the significantly differential expressions (Experimental group: unfertilized egg samples, control group: female adult samples).The blue dots represent down-regulated differentially expressed genes, the red dots represent up-regulated differentially expressed genes, and the gray dots represent non-differentially expressed genes.

Table 1
Summary statistics of female adult and unfertilized egg of S. furcifera transcriptome sequencing and assembly.

Table 2
Annotation result statistics between unigenes and databases.

Table 5
The 65 maternal effect genes of S. furcifera.