Characterization and expression analysis of genes encoding three small heat shock proteins in the oriental armyworm, Mythimna separata (Walker)

Small heat shock proteins (sHsps) function in the response of insects to abiotic stress; however, their role in response to biotic stress has been under-investigated. Mythimna separata, the oriental armyworm, is polyphenetic and exhibits gregarious and solitary phases in response to high and low population density, respectively. In this study, three genes were identified encoding sHsps, namely MsHsp19.7, MsHsp19.8 and MsHsp21.4, and expression levels in solitary and gregarious M. separata were compared. The deduced protein sequences of the three MsHsps had molecular weights of 19.7, 19.8 and 21.4 kDa, respectively, and contained a conserved α-crystalline domain. Real-time PCR analyses revealed that the three sHsps were transcribed in all developmental stages and were dramatically up-regulated at the 6th larval stage in gregarious individuals. Expression of the three MsHsps was variable in different tissues of 6th instar larvae, but exhibited consistent up- and down-regulation in the hindgut and Malpighian tubules of gregarious individuals, respectively. In addition, MsHsp19.7 and MsHsp19.8 were significantly induced when solitary forms were subjected to crowding for 36 h, but all three MsHsps were down-regulated when gregarious forms were isolated. Our findings suggest that population density functions as a stress factor and impacts MsHsps expression in M. separata.


Introduction
High population density (crowding) is a complex stress that impacts the morphology, behavior, life history and physiology of insects [1][2][3] and their population dynamics in the field [4]. To overcome the unfavorable effects of crowding, insects skillfully adopt one or more strategies. For example, insects may alter their phenotype or behavior to adapt to crowding or they may reallocate resources normally used for basic functions (e.g. development, reproduction, nutrient assimilation, and immunity) to cope with changes in population density [5][6][7]. Phase polyphenism is a phenotypic adaption to crowding that has been observed in Orthopterans, Lepidopterans, Hemipterans and Coleopterans [6,[8][9][10]. Solitary  been observed in selected species when subjected to low and high population density, respectively. Gregarious individuals are typically characterized by darker or more melanized cuticles than that of solitary forms [11]. Furthermore, variations in morphology, behavior, life history and disease resistance have been reported in the two insect phases [1,[11][12][13][14][15]. Heat shock proteins (Hsps) are biosynthesized in response to a variety of stressors. As molecular chaperones, Hsps perform critical functions in protein folding, assembly, degradation, and intracellular localization under hospitable and inhospitable conditions [16][17][18]. Insect Hsps can be classified into four general families, e.g. Hsp90, Hsp70, Hsp60, or small Hsps (sHsps); these families are named according to protein size and structural characteristics [19]. sHsp family members exhibit high diversity due to variability in function, structure, and size   [20,21]. sHsps usually prevent protein aggregation and facilitate the correct refolding of denatured proteins under diverse stressful conditions, such as heat, cold, oxidation, drought, UV radiation, hypertonic stress and chemical exposure [19]. Apart from the stress response, some sHsps also function in insect metamorphosis and development [22][23][24][25], longevity [26] and diapause [27][28][29][30]. Recently, some studies have reported that sHsps are also involved in immune responses when insects are colonized by infectious microorganisms [27,31]. However, studies on sHsps have largely focused on model insects and sHsp roles in response to abiotic stress, including extreme temperature, UV irradiation, oxidation, chemicals expsoure, etc. Little is known about sHsp functions in response to biotic stressors such as variations in population density.
Mythimna separata (Walker), which is commonly known as the oriental armyworm, is a formidable pest in Asia. M. separata exhibits polyphenism with solitary and gregarious phases occurring at low and high density, respectively [32], which provides an ideal model to investigate if population density functions as a stressor that impacts organismal physiology [33]. Although the up-regulation of Hsc70 has been observed in gregarious M. separata larvae [34], it is not clear how other Hsps respond to alterations in population density. In this report, we investigate whether the sHsp genes, MsHsp19.7, MsHsp19. 8 and MsHsp21.4, are up-regulated by alterations in M. separata population density and if variability occurs among gregarious and solitary phases. Our findings provide some understanding of the ecological impact of sHsp expression in the evolution and adaptation of M. separata.

Ethics statement
The M. separata larvae were collected from corn stalks cultivated in Qianxi county, Guizhou province (27˚01 0 39.72@N, 106˚20 0 2.92@E), in 2015. In present study, there were no specific permits being required for the insect collection. No endangered or protected species were involved in the field studies. The ''List of Protected Animals in China" does not contain the M. separata which are common insect.

Preparation of samples
Developmental stages (e.g. eggs, 1 st -6 th instar larvae, pupae, and adults) and tissues of 6 th instar larvae (heads, epidermis, foregut, midgut, hindgut and Malpighian tubules) were collected from solitary and gregarious insects as described [32], frozen in liquid nitrogen, and stored at -80˚C until analysis.
The impact of crowding and isolation were evaluated by crowding solitary forms of M. separata and isolating gregarious M. separata, respectively. Sixth instar larvae of solitary M. separata were subjected to crowding by grouping 40 individuals in a 1 L cylinder; conditions for isolation involved separating 6 th instar larvae of gregarious M. separata and placing them in individual 300 mL plastic cylinders [32]. Following treatment, the samples were collected and profiles were examined for expression of the three sHsp genes. Treatments consisted of three larvae and were replicated three times.

RNA isolation, cDNA synthesis and RT-PCR
The SV Total RN A isolation system was used to extract total RNA as recommended (Promega, WI, USA), and DNase I was used to remove residual genomic DNA. RNA quality was evaluated by electrophoresis and UV spectrophotometry as described [32]. The First Strand cDNA Synthesis Kit cDNA was used to generate cDNA from 1 μg total RNA as recommended (Fermentas, Canada), and cDNAs were stored at -20˚C until needed.
Degenerate primers were designed according to the conserved α-crystallin domains of sHsps genes from Noctuidae species, and used to amplify partial sequences of three M.separata sHsp genes by RT-PCR (S1 Table). The reaction conditions for PCR, extraction from agarose gels, cloning, and sequencing followed established protocols [32].
The obtained partial sequences of the three sHsp genes were utilized to design gene-specific primers. Total RNA (1 μg) was used in 5 0 -and 3 0 -RACE with the SMARTer 1 RACE 5'/3' Kit as recommended (Takara Bio USA, Inc.) (S1 Table). RACE was conducted and PCR products were purified, cloned and sequenced as described previously [32]. The initial cDNA and 5 0and 3 0 -RACE products were assembled to obtain full-length cDNA.

Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR was executed using a BioRad CFX96 system (Hercules, CA, USA) in a 20 μL reaction volume containing SsoAdvanced Universal SYBR Green Supermix (10 μL, Bio-Rad), gene-specific primers (1 μL each, S1 Table), cDNA template (1 μL), and ddH 2 O (7 μL). PCR and melting curve analysis were conducted using established parameters [32]. Actin was used to normalize transcript abundance for developmental stage samples, and Tubulin was used to normalize expression for different tissues and population densities [35]. Every treatment contained four replications, and each replication contained triplicate samples.

Statistical analysis
Data were expressed as means ± SE. The comparative Ct method was used to calculate relative expression levels and expressed as 2 −44Ct [36]. Differences between solitary and gregarious phases were discovered using the Student's t-test, and results were considered significant at P<0.05. Data Processing System (DPS) software was used to analyze the results [37].

Phylogenetic analysis of the three MsHsps
Twenty two sHsps, including twenty from Lepidopteran species and two from D.melanogaster, were downloaded from NCBI and maximum likelihood method was used to generate a phylogenetic tree. As shown in Fig 2, MsHsp19.7 and MsHsp19.8 were assigned to a cluster, and separated from MsHsp21.4. Specifically, MsHsp19.7 grouped with HaHsp19.7(Helicoverpa  Table). Nodes were labeled with percent bootstrap values from 2000 re-sampling events, and values less than 50 were deleted.  (Fig 3C). In general, the three sHsp genes were more highly expressed in gregarious 6 th instar larvae as compared to solitary 6 th instar larvae (Fig 3A-3C).

Tissue-specific expression profiles
Tissue-specific expression was analyzed in gregarious and solitary forms of 6 th instar larvae (Fig  4). In HG tissues, the three MsHsps were expressed at 3.07   (Fig 5B, 5D and 5F).

Discussion
In this study, three genes encoding sHsps (MsHsp19.7, MsHsp19. 8 [38] and Grapholitha molesta [39]. This level of phylogenetic diversity may be caused by different rates of sHsp evolution and/or the functional diversity of sHsps in insects.
sHsps are known for regulating insect development. In this study, the three MsHsps were expressed in all developmental stages of solitary and gregarious M. separata, suggesting their importance throughout the M. separata lifespan. Variability in life history, morphology, and behavior has been detected in solitary and gregarious forms of M. separata [40,41]. Contrary to expectation, expression of the three MsHsps was not consistently higher in gregarious individuals from egg to the 5 th larval stage, which was similar to results obtained with locusts [33]. A possible explanation is that the small body size evident in these stages reduces contact between individual insects, thus alleviating the crowding-induced stress response [42]. However, in the 6 th instar larvae of gregarious individuals, expression of the three MsHsps was significantly upregulated, potentially due to the increased competition for resources [32]. Interestingly, up-regulation of the three MsHsps was not observed in gregarious pupae or adults, possibly because this pest undergoes dramatic changes in metamorphosis at these stages and crowding becomes less critical.
The three MsHsps were expressed in tissues of both phases, but showed tissue-specific expression patterns, thus suggesting that MsHsps contribute to normal functioning of the organism [43]. Specifically, expression of MsHsp19.7 and MsHsp19.8 in heads was higher in gregarious versus solitary larvae, suggesting that these two MsHsps may respond to the stress signal(s) produced during crowding. The three MsHsps were also upregulated in the hindgut of gregarious larvae, but showed lower levels of expression in Malpighian tubules (Fig 5). Malpighian tubules and hindgut are known to reabsorb water, salts, and other substances before excretion by the insect [24]. Previous studies have shown that gregarious larvae have higher food consumption than solitary forms [40], which could lead to higher production of toxic byproducts. Therefore, the higher expression levels of MsHsps in gregarious M. separata may be needed to protect the hindgut from injury. It remains unclear why expression of the three MsHsps are upregulated in Malpighian tubules of gregarious M. separata, and further studies are need to address this observation.
Previous reports revealed that alterations in population density could induce Hsp expression [2,32,44,45]. In this study, a significant down-regulation of the three MsHsps was observed in gregarious M. separata exposed to isolation for 36 h; therefore, a reduction in crowding-induced stress in M. separata larvae correlated with a decline in population density. In contrast, a dramatic upregulation of MsHsp19.7 and MsHsp19.8 was observed in solitary M. separata after crowding for 36 h, which was similar to reported studies in locusts [33,45].

PLOS ONE
Three small heat shock protein genes in Mythimna separata (Walker) However, the expression of MsHsp21.4 remained unchanged, suggesting that this gene was not induced and/or a longer period may be needed for crowding-induced changes in transcription. Interestingly, recent studies have showed that crowding resulted in down-regulation of sHsps in Drosophila [2], suggesting that sHsp transcription can vary with the organism and its unique response to changes in population density [32].
Prior investigations demonstrated that the upregulation of Hsps had negative physiological impacts [18, 46,47]. Gregarious M. separata generally have smaller body sizes and reduced reproduction as compared to solitary individuals [40,41]. It has been reported that upregulation of small heat shock proteins enhanced resistance to stress in Locusta and Drosophila, but this was accompanied by a decline in reproduction [48,49]. Therefore, a trade-off exists between sHsp production and pupal size and reproduction during crowding. In addition, a faster developmental rate has been also observed in gregarious individuals [40,41], which was likely an environmental adaption to crowding.
Recent reports indicate that gregarious larvae have developed resistance to selected biopesticides [50], which is associated with improved immune system functionality [41,51]. In insects, sHsps play an important role in the immune response [27,31]. Therefore, these studies promote our hypothesis that the upregulation of sHsps in gregarious M. separata may contribute to improved resistance to biopesticides and pathogens. Newly developed technologies, such as RNAi and CRISPR-Cas9 are needed to confirm this hypothesis in future studies. The three MsHsps identified herein may ultimately provide new molecular targets for managing M. separata during crowding.

Conclusion
In summary, three genes encoding small heat shock protein (sHsps) were successfully characterized in M. separata. Expression analysis by qRT-PCR showed that the three MsHsps exhibited variable expression profiles in gregarious and solitary individuals. Moreover, alterations in population density caused large changes in MsHsp expression. Our findings show that MsHsps function in stress-induced changes that arise due to variations in population density. These findings provide valuable information on the roles of MsHsps in M. separata populations undergoing fluctuations in population density.
Supporting information S1