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Two species of Neoperla from Guizhou Province, China, N. bituberculata Du, 2000 and N. dashahena Du, 2005 were described with brief morphological descriptions available only in Chinese and original illustrations being somewhat blurry. Recently, we examined type material of these two species and re-described them with clear colour pictures for the first time.In this paper, detailed English descriptions and colour pictures of Neoperla bituberculata and N. dashahena are provided for the first time. The type locality of N. mnong Stark is from Vietnam and its geographical distribution is also discussed. Additionally, we also recorded the distribution of N. mnong Stark, 1987 in Guizhou, Hunan and Jiangxi Provinces of China for the first time and provided a geographical distribution map of this species.

Abstract Comprehending the phylogeography of invasive organisms enhances our insight into their distribution dynamics, which is instrumental for the development of effective prevention and management strategies. In China, Pomacea canaliculata and Pomacea maculata are the two most widespread and damaging species of the non-native Pomacea spp.. Given this species’ rapid spread throughout country, it is urgent to investigate the genetic diversity and structure of its different geographic populations, a task undertaken in the current study using the COI and ITS1 mitochondrial and ribosomal DNA genes, respectively. The result of this study, based on a nationwide systematic survey, a collection of Pomacea spp., and the identification of cryptic species, showed that there is a degree of genetic diversity and differentiation in P. canaliculata, and that all of its variations are mainly due to differences between individuals within different geographical populations. Indeed, this species contains multiple haplotypes, but none of them form a systematic geographical population structure. Furthermore, the COI gene exhibits higher genetic diversity than the ITS1 gene. Our study further clarifies the invasive pathways and dispersal patterns of P. canaliculata in China to provide a theoretical basis.

The family Nemouridae, belonging to Plecoptera, comprises 21 genera and over 700 species found globally, with the greatest diversity observed in temperate regions. Nemoura Latreille, 1796 and Amphinemura Ris, 1902 are two largest genera of the family with the highest diversity in China.Two new species of Nemoura Latreille, 1796 of the family Nemouridae, Nemoura exterclava Zhu, Rehman & Du sp. nov. and Nemoura cerciserrata Zhu, Du & Rehman sp. nov., are described and illustrated from the Nanling Mountains Region in Guangdong Province, southern China. The morphological characteristics of the new species are compared with related taxa. Additionally, the status of Indonemoura voluta Li & Yang, 2008, originally from Maoershan National Natural Reserve in Guangxi Zhuang Autonomous Region, was addressed and moved to the genus Amphinemura Ris, 1902 on the basis of newly-caught topotypes.

Hainan Province is an island in the south of China and belongs to the Oriental Region. It has a unique geographical location and superior climatic conditions, providing a good living environment for Leuctridae insects. However, the species richness of the stonefly family Leuctridae in Hainan is low. Two species in total have been recorded, Rhopalopsole bawanglinga Li, Li & Yang, 2023 and Rhopalopsole hainana Li & Yang, 2010.A new species of Leuctridae (Plecoptera) from Wuzhi Mountains, Hainan Province of south China, Rhopalopsole wuzhishana sp. nov. is described and illustrated. We summarised the Leuctridae in Hainan Province and provide supplemental description and colour plates of Rhopalopsole hainana Li & Yang, 2010.

Environmental heterogeneity partly drives microbial succession in arthropods, while the microbial assembly mechanisms during environmental changes remain largely unknown. Here, we explored the temporal dynamics and assembly mechanisms within both bacterial and fungal communities in Liriomyza huidobrensis (Blanchard) during the transition from field to laboratory conditions. We observed a decrease in bacterial diversity and complexity of bacterial-fungal co-occurrence networks in leaf miners transitioning from wild to captive environments. Both neutral and null models revealed that stochastic processes, particularly drift (contributing over 70%), play a crucial role in governing bacterial and fungal community assembly. The relative contribution of ecological processes such as dispersal, drift, and selection varied among leaf miners transitioning from wild to captive states. Furthermore, we propose a hypothetical scenario for the assembly and succession of microbial communities in the leaf miner during the short- and long-term transition from the wild to captivity. Our findings suggest that environmental heterogeneity determines the ecological processes governing bacterial and fungal community assembly in leaf miners, offering new insights into microbiome and mycobiome assembly mechanisms in invasive pests amidst environmental change.

The genus Illiesonemoura Baumann, 1975 (Plecoptera, Nemouridae) is a small-sized stonefly with slender and curved embranous cerci. Currently, 18 species of the genus are known worldwide, mainly distributed in the Palaearctic and Oriental Regions, with a total of two species known to China.Three new species of Illiesonemoura Baumann, 1975, I. bituberculata Wang & Du, sp. nov., I. motuoensis Du & Ji, sp. nov. and I. weii Du & Ji, sp. nov. are described and illustrated, based on male adults from China. Illiesonemoura bituberculata is characterised by two pairs of tubercles arising posteromedially from tergum 10 and by two rows of spinules outlining the lateral edge of the ventral sclerite of the epiproct. Illiesonemoura motuoensis is characterised by the heart-shaped epiproct with a thin and slightly sclerotised protrusion between the sclerotised bands. Illiesonemoura weii is characterised by a pair of small knobs on tergum 10, outer lobes of paraprocts basally broad, then slender towards apices with a pointed tip and the epiproct with lateral spinules.

Inhibitor of apoptosis proteins (IAPs) are crucial components of apoptosis that perform vital roles in the regulation of caspase activity in organisms. In this study, two IAPs genes were identified from Cotesia chilonis, the dominant parasitic wasp of Chilo suppressalis. CcIAP1 gene is a typical IAP and contains two BIR domains and a RING domain, whereas CcIAP gene is an atypical IAP1 only containing two BIR domains. Phylogenetic analysis indicated that CcIAP1 and CcIAP were grouped with other Hymenopteran IAPs and IAP1 in C. suppressalis. Real-time quantitative PCR revealed that CcIAP1 and CcIAP genes were both highly induced at −6°C and 30°C, and expression was highest at the third instar stage. The expression of CcIAP1 and CcIAP genes were significantly induced during parasitism of C. suppressalis, and the 7-d time point resulted in the highest expression levels for both genes, in which was an advanced stage of larval development of C. chilonis. RNAi experiments showed that CcIAP1 gene was the key IAP in the regulation of apoptosis of C. chilonis and its host. In conclusion, CcIAP1 and CcIAP correlate with the development of C. chilonis and their responses to temperature stress.

An investigation report of stonefly fauna in Benxi Manchu Autonomous County, Liaoning Province, northeast China (used to be called Manchuria, now includes Liaoning, Jilin, Heilongjiang Provinces and parts of Inner Mongolia, which are adjacent to the Russian Far East and the Korean Peninsula). Materials were studied with field observation in 2018 and 2019.This paper records five families, nine genera and 14 species of stoneflies from Taizi River, Liaoning Province. Nine species have been recorded for the first time in China and the biology of several common species is also reported for the first time.

Abstract The primary function of heat shock transcription factor (HSF) in the heat shock response is to activate the transcription of genes encoding heat shock proteins (HSPs). The phloem-feeding insect Bemisia tabaci (Gennadius) is an important pest of cotton, vegetables and ornamentals that transmits several plant viruses and causes enormous agricultural losses. In this study, the gene encoding HSF (Bthsf1) was characterized in MED B. tabaci. The full-length cDNA encoded a protein of 652 amino acids with an isoelectric point of 5.55. The BtHSF1 deduced amino acid sequence showed strong similarity to HSF in other insects. Expression analyses using quantitative real-time PCR indicated that Bthsf1 was significantly up-regulated in B. tabaci adults and pupae during thermal stress. Although Bthsf1 was induced by both hot and cold stress, the amplitude of expression was greater in the former. Bthsf1 had distinct, significant differences in expression pattern during different duration of high but not low temperature stress. Oral ingestion of dsBthsf1 repressed the expression of Bthsf1 and four heat shock proteins (Bthsp90, Bthsp70-3, Bthsp20 and Bthsp19.5) in MED B. tabaci during hot and cold stress. In conclusion, our results show that Bthsf1 is differentially expressed during high and low temperature stress and regulates the transcription of multiple hsps in MED B. tabaci.

Wuyi Mountains are located in the northern Oriental Region and the edge of the southern Palaearctic Region. They have a unique geographical location, complex landform and superior climatic conditions, providing a good ecological environment for Leuctridae species. However, due to the damage of some holotypes in the 20th century, limited drawings and lack of colour figures, it is necessary to reorganise and supplement the preserved Leuctridae specimens from Wuyi Mountains.In this study, we found that there are twelve species of Leuctridae recorded in Wuyi Mountains, accounting for about 20% of the recorded species of Leuctridae in China. These records include two genera and five new distribution records species: one species of the genus Paraleuctra Hanson, 1941: Paraleuctra orientalis (Chu 1928) and eleven species of the genus Rhopalopsole Klapálek, 1912, including five new distribution records to Wuyi Mountains: Rhopalopsole fengyangshanensis Yang, Shi & Li, 2009; Rhopalopsole sinensis Yang & Yang, 1993; Rhopalopsole yangdingi Sivec & Harper, 2008; Rhopalopsole flata Yang & Yang, 1995; Rhopalopsole basinigra Yang & Yang, 1995. Now a total of twelve species of Leuctridae have been recorded from Wuyi Mountains, Fujian Province of south-eastern China. In this paper, we also provide a key to the male, new images and some notes of these twelve species, except Rhopalopsole recurvispina (Wu, 1949) and Rhopalopsole spiniplatta (Wu, 1949). We failed to collect these two species and we regard R. recurvispina as a nomen dubium, because there are no distinctive features that can be used to distinguish this species.

The genus Sweltsa is a small to medium-sized stonefly with distinct coloured wings, giving the species the common name of green stoneflies. It belongs to the family Chloroperlidae. This genus includes more than 55 species world wide, 14 of which have been reported from China.A new species of the genus Sweltsa Ricker, 1943, Sweltsa liupanshana Rehman, Du & Huo sp. nov. from Ningxia Hui Autonomous Region, Liupan Mountain, China is described; this is the second record of Sweltsa from Ningxia Hui Autonomous Region. In addition, the first female description and male supplementary description of Sweltsa hamula Chen & Du, 2017 from Sichuan Province are provided. Diagnosis, description and colour illustration of the new species and of Sweltsa hamula are provided and the morphological characteristics are compared with closely-related species.

Sweltsa is a genus of green stoneflies in the family Chloroperlidae and is distributed throughout the Nearctic and East Palaearctic Regions. As they are sensitive to pollutants, they are often used as an indicator species for determining the quality of water bodies. There are around 57 species of this genus worldwide and 11 of those have been identified from China.A new species of the Alloperlini genus Sweltsa Ricker, 1943, Sweltsa ligula Rehman, Huo & Du sp. n. is described from Kuankuoshui National Natural Reserve, Suiyang County, Guizhou Province, southwest China. This is the first report of the family Chloroperlidae from Guizhou Province. Diagnosis, description of male, female and nymph, illustration of terminalia and similarities with closely-related species are provided and discussed.

Abstract Deterministic and stochastic forces both drive microbiota assembly in animals, yet their relative contribution remains elusive, especially in wild aquatic-insect-associated fungal communities. Here, we applied amplicon sequencing to survey the assembly mechanisms of the fungal community in 155 wild stonefly individuals involving 44 species of 20 genera within eight families collected from multiple locations in China. Analysis showed that fungal diversity and network complexity differed significantly among the eight stonefly families, and that the fungal communities in stoneflies exhibited a significant distance-decay pattern across large spatial scales. Both a structural equation model and variance partitioning analysis revealed that environmental factors (e.g., geographical, climatic) outweigh host attributes in shaping the fungal community of stoneflies. Using neutral and null model analyses, we also find that deterministic processes play a larger role than stochasticity in driving the fungal community assembly. However, the relative contribution of ecological processes including dispersal, drift, and selection, varied strongly with host taxonomy. Furthermore, environmental conditions also significantly affect the strength of these ecological processes. Overall, our findings illustrate that variations in host attributes and environment factors may moderate the relative influence of deterministic and stochastic processes to fungal community composition in wild stoneflies, which provides new insights into mechanisms of microbial community assembly in aquatic arthropods.

A new species of the genus Suwallia Ricker, 1943 (Plecoptera, Chloroperlidae), Suwallia dengba sp. nov., is described from Tibet and Yunnan, southwestern China. A diagnosis and description of the adult habitus and aedeagal structure are illustrated with color images. Similarities in the terminalia with closely related species are discussed. In addition, an updated key to adult males of the Suwallia species of China is provided.

Abstract Clarifying the mechanisms underlying microbial community assembly from regional microbial pools is a central issue of microbial ecology, but remains largely unexplored. Here, we investigated the gut bacterial and fungal microbiome assembly processes and potential sources in Drosophila simulans and Dicranocephalus wallichii bowringi, two wild, sympatric insect species that share a common diet of waxberry. While some convergence was observed, the diversity, composition, and network structure of the gut microbiota significantly differed between these two host species. Null model analyses revealed that stochastic processes (e.g., drift, dispersal limitation) play a principal role in determining gut microbiota from both hosts. However, the strength of each ecological process varied with the host species. Furthermore, the source‐tracking analysis showed that only a minority of gut microbiota within D. simulans and D. wallichii bowringi are drawn from a regional microbial pool from waxberries, leaves, or soil. Results from function prediction implied that host species‐specific gut microbiota might arise partly through host functional requirement and specific selection across host–microbiota coevolution. In conclusion, our findings uncover the importance of community assembly processes over regional microbial pools in shaping sympatric insect gut microbiome structure and function.

Capniidae are a family of stoneflies, also known as snow flies, who emerge in winter. The phylogeny of Capniidae is widely accepted to be based on morphological analysis. Until now, only five Capniidae mitochondrial genomes have been sequenced so far. In addition, sampling is required to determine an accurate phylogenetic association because the generic classification of this family is still controversial and needs to be investigated further. In this study, the first mitogenome of genus Isocapnia was sequenced with a length of 16,200 bp and contained 37 genes, including a control region, two rRNAs, 22 tRNAs, and 13 PCGs, respectively. Twelve PCGs originated with the common start codon ATN (ATG, ATA, or ATT), while nad5 used GTG. Eleven PCGs had TAN (TAA or TAG) as their last codon; however, cox1 and nad5 had T as their final codon due to a shortened termination codon. All tRNA genes demonstrated the cloverleaf structure, which is distinctive for metazoans excluding the tRNASer1 (AGN) that missed the dihydrouridine arm. A Phylogenetic analysis of the superfamily Nemouroidea was constructed using thirteen PCGs from 32 formerly sequenced Plecoptera species. The Bayesian inference and maximum likelihood phylogeny tree structures derived similar results across the thirteen PCGs. Our findings strongly supported Leuctridae + ((Capniidae + Taeniopterygidae) + (Nemouridae + Notonemouridae)). Ultimately, the best well-supported generic phylogenetic relationship within Capniidae is as follows; (Isocapnia + (Capnia + Zwicknia) + (Apteroperla + Mesocapnia)). These findings will enable us to better understand the evolutionary relationships within the superfamily Nemouroidea and the generic classification and mitogenome structure of the family Capniidae.

ABSTRACT The microbiota of invasive animal species may be pivotal to their adaptation and spread, yet the processes driving the assembly and potential sources of host-microbiota remain poorly understood. Here, we characterized microbiota of four Liriomyza leaf miner fly species totaling 310 individuals across 43 geographical populations in China and assessed whether the microbiota of the wild leaf miner was acquired from the soil microbiota or the host plant microbiota, using high-throughput 16S rRNA sequencing. Bacterial communities differed significantly among four leaf miner species but did not mirror host phylogeny. Microbiota diversity in the native L. chinensis was significantly higher than in three invasive leaf miners (i.e., L. trifolii, L. huidobrensis, and L. sativae), yet the microbial community of the invasive species exhibited a more connected and complex network structure. Structural equation models revealed that host species identity was more important than environmental factors (e.g., geography, climate, or plants) in shaping microbiota composition. Using neutral and null model analyses, we found that deterministic processes like variable selection played a primary role in driving microbial community assembly, with some influence by stochastic processes like drift. The relative degree of these processes governing microbiota was likely correlated with host species but independent of either geographical or climatic factors. Finally, source tracking analysis showed that leaf miners might acquire microbes from their host plant rather than the soil. Our results provide a robust assessment of the ecological processes governing bacterial community assembly and potential sources of microbes in invasive leaf miners. IMPORTANCE The invasion of foreign species, including leaf miners, is a major threat to world biota. Host-associated microbiota may facilitate host adaption and expansion in a variety of ways. Thus, understanding the processes that drive leaf miner microbiota assembly is imperative for better management of invasive species. However, how microbial communities assemble during the leaf miner invasions and how predictable the processes remain unexplored. This work quantitatively deciphers the relative importance of deterministic process and stochastic process in governing the assembly of four leaf miner microbiotas and identifies potential sources of leaf miner-colonizing microbes from the soil-plant-leaf miner continuum. Our study provides new insights into the mechanisms underlying the drive of leaf miner microbiota assembly.

The ability of insets to react efficiently to fluctuation in temperature is crucial for them to survive in variable surroundings. Rapid cold hardening (RCH) is a process that increase cold tolerance in most insect species. The molecular mechanisms of RCH remain largely unknown, and whether it is associated with transcriptional changes is unclear. In this study, we compared the transcriptomes of Liriomyza trifolii and L. sativae exposed to RCH to investigate the transcript abundance due to RCH in both species. RNA-seq revealed 93,166 assembled unigenes, and 34,303 of these were annotated in the L. trifolii and L. sativae transcriptome libraries. After a 4-h treatment at 1°C (RCH) compared with control, 268 and 606 unigenes were differentially expressed in L. trifolii and L. sativae, respectively. When comparing pupae exposed to 2h cold shock directly with pupae went through 4h acclimation prior to 2h cold shock, 60 and 399 unigenes were differentially expressed in L trifolii and L sativae, respectively. Genes that were commonly expressed in both L. trifolii and L. sativae, included cytochrome P450, cuticular protein, glucose dehydrogenase, solute carrier family 22 and cationic amino acid transporter. Additionally, several pathways including galactose metabolism and peroxisome were significantly enriched during RCH. Our results show that the transcriptional response is correlated with RCH in the pupal stage of the two Liriomyza species, but more transcriptional changes were identified in L sativae than in L. trifolii.

Small heat shock proteins (sHSPs) are a very complex protein superfamily that increase insect temperature tolerance. In order to deeply understand the function and role of sHSPs in Chilo suppressalis (Walker), this study isolated and identified two CsHSP genes lacking introns from C. suppressalis, Cshsp23.9 and Cshsp27.3. The cDNA full-length of Cshsp23.9 and Cshsp27.3 were 909 and 1 036 bp encoding 220 and 242 amino acids, respectively. Alignment with homologs and phylogenetic analysis indicated Cshsp23.9 and Cshsp27.3 were two new types of Cshsps in C. suppressalis. Real-time quantitative PCR (qPCR) revealed that Cshsp23.9 had the highest relative expression in hindgut compared with other tissues (head, epidermis, foregut, midgut, fat body, Malpighian tubules, and hemocytes), while Cshsp27.3 expressed the highest in fat body content. When exposed to thermal stress from −11 to 43°C for 2 h, two genes showed different expression patterns. Cshsp23.9 did not respond to low temperature, but could be up-regulated by high temperature and the highest expression temperature was at 36°C. Cshsp27.3 could only be induced by mild temperature, with the highest expression at 15 and 30°C. In conclusion, Cshsp23.9 and Cshsp27.3 existed in different tissues/organs of C. suppressalis, and played different important roles in C. suppressalis to resist temperature stress and regulate physiological activities.

Currently, information on the biology of Plecoptera from China is scarce, particularly on mating behavior. In this paper, the existence of mating mistakes (erroneous mating attempts) involving 13 Chinese stonefly species (belonging to nine genera and three families) is reported. These erroneous mating behaviors can be included into three different categories: mating attempts between conspecific males (including the formation of erroneous mating balls), mating attempts between different taxa (including displacement attempts during copulation), and mating-related behaviors with non-living objects. From these behaviors, some aspects of stoneflies during mating, such as the physical competition between males, the sensorial mechanisms implied in triggering a mating behavior, the conditions favoring the mating mistakes, and the possible consequences of interspecific mating in the hybrid production, are discussed.

The type species of Filchneria Klapálek, 1908, F. mongolica (Klapálek, 1901), is based on a single female collected from Mongolia, but it was considered the same as another species, F. songi from Qinling, China, when the genus Filchneria was proposed. This study narrates the story of these two species, which have been confused for a century. Until now, the distribution of F. mongolica has been confirmed only in Mongolia and Russia, and we recently recorded it for the first time in Inner Mongolia as a new species record in China. Additionally, the genus Sinoperlodes is a junior synonym of Filchneria, as demonstrated by both the morphological and molecular analysis. Phylogenetic analysis based on the subfamily Perlodinae is provided, along with morphological and biogeographic comparisons of Filchneria and its relatives.

Liriomyza trifolii is an important invasive pest that infects horticultural vegetables, displaying a strong competitive advantage and showing great potential for inflicting harm. Chitin synthase is one of the key enzymes in insect chitin metabolism and plays an important role in insect growth and development. In this study, a chitin synthase (CHS) transcript of L. trifolii was cloned, and the results showed that LtCHS belongs to the CHS2 family. The expression analysis indicated the presence of the highest abundance of LtCHS2 in the pupae at different developmental stages but showed no significant difference among different tissues in the adult. Furthermore, a dsRNA immersion method was developed for RNA interference (RNAi) in L. trifolii using LtCHS2 transcript. RNAi can significantly reduce the expression of LtCHS2 in pupae, and the emergence rate of the pupae was significantly lower than that of the control. The results provide a theoretical basis for exploring the role of chitin synthase gene in L. trifolii and proposing new pest control strategies.

Frankliniella occidentalis is an invasive insect pest that incites damage to ornamental and agronomic crops on a global scale. In this study, the effects of temperature on gene expression and enzyme activity were studied for superoxide dismutase (SOD), peroxidase (POD), and glutathione-S-transferase (GST) in F. occidentalis. SOD, POD and GST enzyme activity increased significantly at 35–37 °C but declined as the temperature increased to 41 °C. In a time course study at 35 °C, SOD, POD and GST activities were significantly elevated at 0.5, 1 and 2 h in comparison to the control at 26 °C. Expression patterns were evaluated for the three antioxidant genes under high and low temperature stress. In a time course study at –4 °C, SOD, POD and GST expression peaked at 1 h and declined at 2 h of exposure. In contrast, when transcription was monitored at 35 °C, expression was lowest at 1 h and increased at 2 h. The results provide data that will be useful in deciphering the role of antioxidant enzymes in the adaptation of F. occidentalis to climate change.

Liriomyza trifolii is an invasive pest of horticultural and vegetable crops that possesses robust competitive advantages that enable it to replace closely-related species. High temperatures often occur concomitantly with insecticide usage during L. trifolii outbreaks. In this study, we compared the transcriptomes of L. trifolii exposed to high temperature (40 °C T40), insecticide (LC50 of technical grade abamectin, I50) and combined high temperature and abamectin exposure (IT5040, I50 followed by T40; and TI4050, T40 followed by I50). RNA-seq generated and revealed 44,633 unigenes with annotation data; these were compared with COG and KEGG databases for functional classification and enrichment analysis. Compared with the I50 treatment, COG classification indicated that ‘post-translational modification, protein turnover, chaperones’ was enriched in the IT5040 treatment. In the TI4050 treatment, ‘carbohydrate transport and metabolism’ was the most abundant group. The most enriched KEGG pathways in the TI4050 and IT5040 treatments were ‘longevity regulating pathway - multiple species’ and ‘protein processing in endoplasmic reticulum’, respectively. Subsequent annotation and enrichment analyses indicated that stress-related genes such as CYP450s and HSPs were differentially expressed in the I50 vs. TI4050 or I50 vs. IT5040 treatment groups. Three commercial insecticide formulations were also used to further verify the expression of selected differentially-expressed genes. This study will be conductive to consider the temperature effect on insecticide tolerance in L. trifolii, and provides a framework for improving the application efficiency of insecticides in hot weather, which will ultimately reduce the overuse of pesticides.

The leafminer fly, Liriomyza trifolii, is an invasive pest of horticultural and vegetable crops that possesses a robust competitive ability when compared to congeneric species, especially with respect to temperature and insecticide tolerance. Abamectin, which is commonly used to control L. trifolii in the field, was selected as the target insecticide in this study. Our objective was to study the effect of abamectin and high temperature stress on L. trifolii mortality and the expression of genes encoding cytochrome P450 (CYP450s) and heat shock proteins (Hsps) by quantitative real-time reverse transcriptase PCR (qRT-PCR). When L. trifolii was exposed to abamectin followed by exposure to 40 °C (LC50 +HT40), mortality showed a significant increase, whereas exposure to 40 ℃ followed by abamectin (HT40+LC50) reduced mortality relative to abamectin or HT40 alone. Expression of three CYP450s in the CYP4 family was highest in the HT40+LC50 treatment, followed by the LC50+HT40 treatment. The expression levels of CYP18A1 (CYP18 family) were not significantly different among treatments, and CYP301A1 (CYP301 family) was only sensitive to temperature (HT40). The expression of five sHsps showed similar expression patterns and were highly responsive to the LC50+HT40 treatment, followed by the HT40 and HT40+LC50 treatments. Based on CYP450s and Hsps expression levels, our findings that suggest that L. trifolii exhibits adaptive cross-tolerance to high temperature and abamectin. This study provides a framework for selecting the most effective application time for abamectin with respect to controlling L. trifolii, which will ultimately reduce the overuse of pesticides.

Cotesia chilonis (Munakata) is the dominant parasitic wasp of the rice pest, Chilo suppressalis (Walker), and is a valuable parasitic wasp for the prevention and control of C. suppressalis. In this study, developmental indicators and expression of Cchsp11.0 (heat shock protein 11.0) and Cchsf (heat shock factor) were compared for C. chilonis at 27 °C and 36 °C. Developmental duration, morphology, emergence rate, and number of C. chilonis offspring were shortened at 36 °C while the ratio of females to males increased. Cchsp11.0 and Cchsf were highly expressed in the 1st instar stage at 36 °C, and Cchsp11.0 expression gradually decreased as C. chilonis matured; Cchsf expression was not correlated with Cchsp11.0 expression. Compared with 27 °C, the expression pattern of Cchsp11.0 and Cchsf was also not consistent, and Cchsp11.0 expression increased significantly at the adult stage. In conclusion, mildly high temperatures impact growth, development and reproduction of C. chilonis and stimulate the expression of Cchsp11.0 and Cchsf, and Cchsp11.0 and Cchsf play different roles in different developmental stages of C. chilonis at normal and high temperature.

Wolbachia are widespread maternally-inherited endosymbiotic bacteria that infect numerous arthropods. This study represents a thorough survey of the Wolbachia infection patterns in the pink stem borer, Sesamia inferens (Walker), an important rice pest in China, based on nucleotide comparisons for the surface protein (wsp) and cell division protein (ftsZ) genes. The effects of Wolbachia on mtDNA variation and evolution of S. inferens were also investigated. Although we identified six genetically diverse strains, we found infections to be infrequent, with only 7.8% of hosts infected, and identified geographical differences in infection rates between southern and northern populations. Nucleotide indexes (haplotype diversity (Hd), nucleotide diversity (Π) and number of variable sites (S) of mtDNA in infected populations were not significantly lower or higher than that in the uninfected populations. Furthermore, there was no association between Wolbachia infection status and phylogeny of mtDNA haplotypes. Analysis of molecular variance (AMOVA) showed that significant differentiation mainly existed within groups rather than among the groups. Additionally, using Tajima's D and Fu's F values, the mtDNA genes did not deviate significantly from neutral evolution. Taken together these results indicate that currently there were no effects of Wolbachia infection on host mtDNA variation and evolution in S. inferens.

We recently examined specimens of the genus Paraleuctra Hanson, 1941, from Zhejiang Province and Sichuan Province, China, and two new synonyms are established based on morphological and molecular data. Paraleuctra sinica Yang and Yang, 1995, and Paraleuctra tianmushana Li and Yang, 2010, are synonymized with Paraleuctra orientalis (Chu, 1928). Additionally, we provide new images of Paraleuctra cervicornis Du and Qian, 2012, to facilitate identification of this species.

Due to varietal differences, diminutive size, and similar morphological characters, it is difficult to classify and identify Liriomyza spp., a genus comprised of economically-important, highly-polyphagous insect pests. In this study, we reconfirmed the morphological characteristics of three closely-related invasive leafminers, L. trifolii, L. sativae, and L. huidobrensis. Morphological results showed that characteristics imparted by the male genitalia were the most reliable morphological features for identification. The colors exhibited by vertical setae were variable among species, and the ratio of the length of the ultimate section of vein CuA1 divided by penultimate section also varied within species. Although the patterns of abdominal tergites were diverse among Liriomyza spp., L. trifolii exhibited a unique pattern with a yellow patch at the 5th black visible tergite; this pattern can be profiled as a prominent characteristic for morphological identification. In order to identify the three Liriomyza spp. quickly and accurately, we developed an improved molecular identification method using multiplex PCR based on the gene encoding mitochondrial cytochrome oxidase I (COI); this method enabled direct identification based on the size of amplified products. The results of this study provide a valuable reference for the identification of Liriomyza spp., which will ultimately improve our ability to control individual species.

Of the roughly 400 species of Perlidae in the world, most species are widely distributed in the northern hemisphere, but a few can be found in South Africa and South America. There are only five species in the genus Flavoperla of the family Perlidae in China. To gain a better understanding of the architecture and evolution of mitochondrial genome in Flavoperla, the entire mitochondrial genome (mitogenome) of a Chinese Flavoperla biocellata Chu, 1929 from family Perlidae (Insecta: Plecoptera) was sequenced. The 15,805-bp long mitochondrial genome of F. biocellata contained 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a putative control region (CR). The gene arrangement of F. biocellata was identical with that of other stoneflies and with the fly Drosophila yakuba. Most PCGs of F. biocellata used the standard ATN start codons and complete TAN termination codons. Twenty-one of the 22 tRNA genes exhibited cloverleaf secondary structures, but the dihydrouridine (DHU) arm of trnSer (AGN) was completely reduced. Phylogenetic analyses with both Bayesian inference (BI) and maximum likelihood methods (ML) generated similar topology, both supporting the monophyly of all stonefly families and the infraorder Systellognatha. The phylogenetic analysis based on mitochondrial genomic data from 30 stonefly species recovered a well-supported tree resolving higher-level relationships within Plecoptera. The northern hemisphere suborder Arctoperlaria divided into two groups, Euholognatha and Systellognatha. The southern hemisphere suborder Antarctoperlaria formed two clades: Eustheniidae+Diamphipnoidae and Austroperlidae+ Gripopterygidae; consistent with relationships proposed based on morphology. The final relationships within Plecoptera were recovered as (((Perlidae+(Perlodidae+Chloroperlidae))+(Pteronarcyidae+(Peltoperlidae+Styloperlidae))) +(Taeniopterygidae+(Capniidae+(Nemouridae+Notonemouridae))))+ (Gripopterygoidae+Eusthenioidae).

Frankliniella occidentalis (Pergande) is an invasive pest that endangers a wide variety of horticultural and agronomic crops. HSP70 is the most important member of the heat shock protein (HSP) family and plays an important role in insect thermal tolerance. In this study, a new gene encoding HSP70 from F. occidentalis, Fohsp706, was selected from the F. occidentalis transcriptome exposed to thermal stress (40 °C) and cloned by RT-PCR and RACE. Further characterization indicated that Fohsp706 localizes to the cytoplasm and does not contain introns. Quantitative real-time reverse transcriptase PCR indicated that Fohsp706 expression was significantly up-regulated by thermal stress; furthermore, there were significant differences in Fohsp706 expression in adults and second instar nymphs after heat stress. Our results indicated that Fohsp706 contributes to thermotolerance in F. occidentalis and provides another example of how this pest adapts to unfavorable environmental conditions.

Heat shock protein 70 (HSP70) is one of the most important members in the heat shock protein family, and plays important roles in the thermotolerance of insect. To explore the molecular mechanism of thermotolerance of Frankliniella occidentalis adults, the difference in the expression of HSP70s in F. occidentalis male or female adults under the thermal stress was studied under the laboratory conditions. Two full length cDNAs of HSP70s gene (Fohsc704 and Fohsc705) were cloned from F. occidentalis by using RT-PCR and RACE. The genomic sequence was demonstrated by genomic validation, and the position and size of the intron were analyzed by sequence analysis of cDNA. Real-time PCR was used to analyze the HSP70 expression patterns. The cDNA of Fohsc704 and Fohsc705 possessed 2 073 and 1 476 bp which encoded 690 and 491 amino acids (aa) with a calculated molecular weight of 75 and 54 kDa, respectively. Four introns in Fohsc704 and six introns in Fohsc705 protein were found. However, the HSP70 protein sequences in our study were ended with EKKN and GIFL, which were different from the reported FoHSP70s. Various expression patterns of Fohsc704 and Fohsc705 were found in both genders of F. occidentalis under thermal stress. The expression of Fohsc704 and Fohsc705 reached to the highest level at –12 and –8°C in male adults, respectively, and Fohsc705 expressed the highest level at 33°C in female adults. In conclusion, HSP70s of F. occidentalis in our study are novel heat shock proteins. There were difference in expression patterns of the two hsc70s in genders of F. occidentalis, and the two HSP70s play important roles in the thermotolerance of F. occidentalis.

Five genes encoding heat shock proteins (HSPs), Cchsp40, Cchsp60, Cchsp70, Cchsc70 and Cchsp90, were cloned and sequenced from Cotesia chilonis using RT-PCR and RACE. The cDNA sequences of Cchsp40, Cchsp60, Cchsp70, Cchsc70 and Cchsp90 were 1 265, 2 551, 2 094, 2 297 and 2 635 bp in length, respectively, with a molecular weight (MW) of 39.1, 60.6, 71.45, 70.19 and 82.92 kDa, respectively. The predicted amino acid sequences of these proteins showed high similarities with published HSPs of other insects in Hymenoptera. Analysis of genomic DNAs indicated that Cchsp40, Cchsp60, Cchsp70 and Cchsp90 lacked introns, but Cchsc70 contained an intron. The results also suggested that CcHSP40 in C. chilonis was the Type II HSP40, CcHSP60 was a member of the mitochondrial HSP60 family, and CcHSP90 was a part of cytoplasmic HSP90A family. Expression patterns varied in the five Cchsps in response to temperature. Expression of Cchsp40 and Cchsp60 was induced significantly by cold but not heat stress. Cchsp70 and Cchsc70 showed similar response to the thermal stress and could be induced by both cold and heat, but their expression levels were consistently lower than that of Cchsp40 and Cchsp60. Cchsp90 could be induced by heat stress and mild cold, but not cold stress. In addition, the results demonstrated Cchsc70 might be constitutive and inducible protein that was expressed during normal cell functioning and also up-regulated in response to stressful stimuli while Cchsp70 was solely inducible protein induced by temperature changes. Overall, results generated from this study could significantly advance the understanding of Cchsps in response to temperature and provide important biological information for C. chilonis insects that reared under different temperatures.

Rapid cold hardening (RCH) is a rapid and critical adaption of insects to sudden temperature changes but is often overlooked or underestimated as a component of survival. Thus, interspecific comparisons of RCH are needed to predict how phenotypes will adapt to temperature variability. RCH not only enhances cold survival but also protects against non-lethal cold injury by preserving essential functions such as locomotion, reproduction, and energy balance. This study investigated the difference in basal cold tolerance and RCH capacity of L. trifolii and L. sativae. In both species, the cold tolerance of pupae was significantly enhanced after short-term exposure to moderately cold temperatures. The effect of RCH last for 4 h in L. sativae but only 2 h in L. trifolii. Interestingly, L. trifolii adults had a RCH response but L. sativae adults failed to acclimate. Short-term acclimation also lowered the supercooling point significantly in the pupae of both species. Based on these results, we propose a hypothesis that these differences will eventually affect their competition in the context of climate change. This study also provides the basis for future metabolomic and transcriptomic studies that may ultimately uncover the underlying mechanisms of RCH and interspecific competition between L. trifolii and L. sativae.

Insects represent a sustainable, protein-rich food source widely consumed in Asia, Africa, and South America. Eating Clanis bilineata tsingtauica Mell is common in the eastern part of China. A comparative characterization of nutrients in the meat and epidermis of C. bilineata tsingtauica was performed in this study. The results showed this insect to be high in nutrients, particularly in the epidermis where protein total was 71.82%. Sixteen different amino acids were quantified in C. bilineata tsingtauica, and the ratio of essential to nonessential amino acids in the epidermis and meat was 68.14% and 59.27%, respectively. The amino acid composition of C. bilineata tsingtauica is balanced, representing a high-quality protein source. Eight minerals were quantified in C. bilineata tsingtauica, including four macro and four trace elements. Fe in the epidermis and Zn in the meat were abundant at 163.82 and 299.31 μg/g DW, respectively. The presence of phytic acid impacted the absorption of mineral elements in food. We also detected phytic acid in C. bilineata tsingtauica. The molar ratio of phytic acid to zinc (PA/Zn) in C. bilineata tsingtauica was very low (3.28) compared to Glycine max and Cryptotympana atrata, which indicated that mineral utilization was high. In conclusion, this study confirms that C. bilineata tsingtauica is a highly nutritious food source for human consumption, and the results provide a basis for further consumption and industrialization of this edible insect.

Small heat shock proteins (sHSPs) are members of the heat shock protein (HSP) family that play an important role in temperature stress, and heat shock factors (HSFs) are transcriptional activators that regulate HSP expression. Cotesia chilonis, the major endoparasitoid of Chilo suppressalis, modulates the C. suppressalis population in the field. In this study, we cloned and characterized two genes from C.chilonis: the heat-induced HSP11.0 gene (Cchsp11.0) that consisted of a 306-bp ORF, and the master regulator HSF (Cchsf) containing an 1875-bp ORF. CcHSP11.0 contained a chaperonin cpn10 signature motif that is conserved in other hymenopteran insects. CcHSF is a typical HSF and contains a DNA-binding domain, two hydrophobic heptad repeat domains, and a C-terminal trans-activation domain. Neither Cchsp11.0 or Cchsf contain introns. Real-time quantitative PCR revealed that Cchsp11.0 and Cchsf were highly induced at 36 °C and 6 °C after a 2-h exposure. Overall, the induction of Cchsf was lower than Cchsp11.0 at low temperatures, whereas the opposite was true at high temperatures. In conclusion, both Cchsp11.0 and Cchsf are sensitive to high and low temperature stress, and the expression pattern of the two genes were positively correlated during temperature stress.

Abstract In this study, we report the complete mitochondrial genome (mitogenome, mtDNA) of Aposthonia borneensis and compare it with another sequenced webspinner, Aposthonia japonica. The A. borneensis mitogenome is smaller than A. japonica, but the size of each gene and the A + T content of protein-coding genes (PCGs) are almost identical in the two mitogenomes. Among the PCGs, atp6 shows the highest evolutionary rate and cox1 the lowest. The mtDNA map in A. borneensis is similar to Drosophila yakuba, but distinctly different from A. japonica, which has extensive rearrangement. Phylogenetic analyses dated the divergence time of the two webspinners at ca. 103 Ma. We speculate that the most recent common ancestor (MRCA) of A. borneensis and A. japonica was divided into several geographic groups during the Pangea breakup. Geographic isolation between the Japanese islands and the continental southeastern Asia resulted in the divergent evolution of A. borneensis and A. japonica, thus generating mtDNA structural variations between the two species. Based on the phylogenetic analyses and specific distributional features, the genus Aposthonia was supported as non-monophyly, and we speculate that both highly rearranged and relatively conserved mitogenomes exist in other webspinners.

A new whitefly species, Aleuromarginatus dielsianae Wang & Xu, sp. n. collected from Millettia dielsiana Harms (Rosales: Fabaceae) in Jiangshan (28°40'N, 118°40'E, 512 m) and Xinchang (29°22'N, 120°46'E, 308 m), Zhejiang, China, is described and illustrated. This new species is characterized by the dark brown lateral margin area and a pair of longitudinal furrows extending from the cephalothorax to the vasiform orifice. The submargin has an elongate-oval fold at the base of each marginal tooth and with 3-4 rows of irregular shaped papillae, nine pairs submedian setae and 13 pairs submarginal setae. Thoracic and caudal tracheal folds and pores discernible. An identification key and checklist of species of Aleuromarginatus known from China are provided.

Abstract Corythucha ciliata (Say), an invasive pest originating from North America, causes severe damage on sycamore trees. However, little is known about the population genetics and evolutionary forces underlying the invasiveness of this important pest. In the present study, we use three mitochondrial genes (COI, ND1 and ND5) and nine microsatellite markers to investigate the population genetics of C. ciliata and retrace its spread through China. The results suggest a low level of genetic diversity in Chinese and European populations of C. ciliata. Our results indicate that populations of C. ciliata have obvious genetic structure, and genetic differentiation is not caused by geographic isolation. In median-joining networks, we observed a higher frequency of shared haplotypes in groups 1 and 3. Based on gene flow and approximate Bayesian computation analyses, we discovered that C. ciliata first invaded the east coast of China and subsequently moved inland. Demographic analysis suggested that populations of C. ciliata in China may have undergone a recent bottleneck effect. Finally, our results suggest that population structure, high gene flow and environmental conditions have favored the broad invasiveness of this important pest.

Small heat shock proteins (sHSPs) are probably the most diverse in structure and function among the various super-families of stress proteins, and they play essential roles in various biological processes. The sweet potato whitefly, Bemisia tabaci (Gennadius), feeds in the phloem, transmits several plant viruses, and is an important pest on cotton, vegetables and ornamentals. In this research, we isolated and characterized three α-crystallin/sHSP family genes (Bthsp19.5, Bthsp19.2, and Bthsp21.3) from Bemisia tabaci. The three cDNAs encoded proteins of 171, 169, and 189 amino acids with calculated molecular weights of 19.5, 19.2, and 21.3 kDa and isoelectric points of 6.1, 6.2, and 6.0, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in Hemiptera and Thysanoptera insects species. All three sHSPs genes from Bemisia tabaci lacked introns. Quantitative real-time PCR analyses revealed that the three BtsHSPs genes were significantly up-regulated in Bemisia tabaci adults and pupae during high temperature stress (39, 41, 43, and 45 °C) but not in response to cold temperature stress (−6, −8, −10, and −12 °C). The expression levels of Bthsp19.2 and Bthsp21.3 in pupae was higher than adults in response to heat stress, while the expression level of Bthsp19.5 in adults was higher than pupae. In conclusion, this research results show that the sHSP genes of Bemisia tabaci had shown differential expression changes under thermal stress.

In quantitative real-time PCR (qRT-PCR), data are normalized using reference genes, which helps to control for internal differences and reduce error among samples. In this study, the expression profiles of eight candidate housekeeping genes, 18S ribosomal (18S rRNA), elongation factor (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein L17 (RPL17), histone 3 (H3), arginine kinase (AK), amd β-Actin (ACTB), were evaluated in the parasitic wasp Cotesia chilonis in response to different temperatures. Specifically, the performance and stabilities of these genes were compared in adult wasps maintained in a growth condition at 27°C (normal storage conditions) and in adults obtained from pupae refrigerated at 4°C for five days (cold storage conditions). Data were analyzed using the ΔCt method, BestKeeper, NormFinder, and geNorm. The optimal numbers and stabilities of reference genes varied between the two temperature treatments (27°C and 4°C). In samples stored at normal developmental temperature (27°C), the requirement for normalization in response to low temperature exposures was three genes (18S, H3, AK), whereas normalization in response to high temperature exposures required only two reference genes (GAPDH, ACTB). In samples stored at cold temperature (4°C), for low temperature exposures two reference genes (RPL17, RPL10) were required for standardization, while following high temperature exposures three reference genes (18S, H3, ACTB) were needed. This study strengthens understanding of the selection of reference genes before qRT-PCR analysis in C. chilonis. The reference genes identified here will facilitate further investigations of the biological characteristics of this important parasitoid.

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

Despite the severe ecological damage and economic loss caused by invasive species, the factors contributing to successful invasion or displacement remain elusive. The whitefly, Bemisia tabaci (Gennadius), is an important invasive agricultural pest worldwide, causing severe damage to numerous crops by feeding or transmitting plant viruses. In this study, we monitored the dynamics of two invasive whitefly cryptic species, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED), in Jiangsu, China, from 2005−2016. We found that B. tabaci MED quickly established and asserted dominance over MEAM1, resulting in their population displacement in Jiangsu in only three years (from 2005 to 2008). We further investigated the possible mechanisms underlying the successful invasion and competitive displacement from a genetic perspective. Based on sequencing of mitochondrial gene sequences from large numbers of whitefly samples, multiple invasion events of MED were validated by our genetic analyses. MED invaded Jiangsu starting from multiple introduction sites with secondary and/or subsequent invasive events. This may favor their invasion and displacement of MEAM1. This study advances our understanding of the mechanisms that enabled the successful invasion of MED.

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex, including members that are pests of global importance. This study presents a screening of B. tabaci species in China for infection by the primary endosymbiont, Portiera aleyrodidarum, and two secondary endosymbionts, Arsenophonus and Cardinium. The results showed that P. aleyrodidarum was detected in all B. tabaci individuals, while Arsenophonus was abundant in indigenous species of B. tabaci Asia II 1, Asia II 3, and China 1 but absent in the invasive species, Middle East-Asia Minor 1 (MEAM1); Cardinium presented in the Mediterranean (MED), Asia II 1 and Asia II 3 species but was rarely detected in the MEAM1 and China 1 species. Moreover, phylogenetic analyses revealed that the P. aleyrodidarum and mitochondrial cytochrome oxidase 1 (mtCO1) phylograms were similar and corresponding with the five distinct cryptic species clades to some extent, probably indicating an ancient infection followed by vertical transmission and subsequent co-evolutionary diversification. In contrast, the phylogenetic trees of Arsenophonus and Cardinium were incongruent with the mtCO1 phylogram, potentially indicating horizontal transmission in B. tabaci cryptic species complex. Taken together, our study showed the distinct infection status of endosymbionts in invasive and indigenous whiteflies; we also most likely indicated the co-evolution of primary endosymbiont and its host as well as the potential horizontal transfer of secondary endosymbionts.

Small heat shock proteins (sHSPs) comprise numerous proteins with diverse structure and function. As molecular chaperones, they play essential roles in various biological processes, especially under thermal stresses. In this study, we identified three sHSP-encoding genes, LtHSP19.5, LtHSP20.8 and LtHSP21.7b from Liriomyza trifolii, an important insect pest of ornamental and vegetable crops worldwide. Putative proteins encoded by these genes all contain a conserved α-crystallin domain that is typical of the sHSP family. Their expression patterns during temperature stresses and at different insect development stages were studied by reverse-transcription quantitative PCR (RT-qPCR). In addition, the expression patterns were compared with those of LtHSP21.3 and LtHSP21.7, two previously published sHSPs. When pupae were exposed to temperatures ranging from −20 to 45 °C for 1 h, all LtsHSPs were strongly induced by either heat or cold stresses, but the magnitude was lower under the low temperature range than high temperatures. Developmentally regulated differential expression was also detected, with pupae and prepupae featuring the highest expression of sHSPs. Results suggest that LtsHSPs play a role in the development of the invasive leaf miner fly and may facilitate insect adaptation to climate change.

Aquaporins are integral membrane proteins some of which form high capacity water-selective channels, promoting water permeation across cell membranes. In this study, we isolated the aquaporin transcript (CsDrip1) of Chilo suppressalis, one of the important rice pests. CsDrip1 included two variants, CsDrip1_v1 and CsDrip1_v2. Although CsDrip1_v2 sequence (>409 bp) was longer than CsDrip1_v1, they possessed the same open reading frame (ORF). Protein structure and topology of CsDrip1 was analyzed using a predicted model, and the results demonstrated the conserved properties of insect water-specific aquaporins, including 6 transmembrane domains, 2 NPA motifs, ar/R constriction region (Phe69, His194, Ser203, and Arg209) and the C-terminal peptide sequence ending in “SYDF.” Our data revealed that the Xenopus oocytes expressing CsDrip1 indicated CsDrip1 could transport water instead of glycerol, trehalose and urea. Further, the transcript of CsDrip1 expressed ubiquitously but differentially in different tissues or organs and developmental stages of C. suppressalis. CsDrip1 mRNA exhibited the highest level of expression within hindgut and the third instar larvae. Regardless of pupae and adults, there were significantly different expression levels of CsDrip1 gene between male and female. Different from at low temperature, the transcript of CsDrip1 in larvae exposed to high temperature was increased significantly. Moreover, the mRNA levels of CsDrip1 in the third instar larvae, the fifth instar larvae, pupae (male and female), and adults (male and female) under different humidities were investigated. However, the mRNA levels of CsDrip1 of only female and male adults were changed remarkably. In conclusions, CsDrip1 plays important roles in maintaining water homeostasis in this important rice pest.

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

Liriomyza trifolii is a highly-invasive leafmining insect that causes significant damage to vegetables and horticultural crops worldwide. Relatively few studies have quantified gene expression in L. trifolii using real-time quantitative PCR (RT-qPCR), which is a reliable and sensitive technique for measuring gene expression. RT-qPCR requires the selection of reference genes to normalize gene expression data and control for internal differences between samples. In this study, nine housekeeping genes from L. trifolii were selected for their suitability in normalizing gene expression using geNorm, Normfinder, BestKeeper, the ΔCt method and RefFinder. HSP21.7, which encodes heat shock protein 21.7, was used as a target gene to validate the expression of candidate reference genes. Results indicated that ACTIN and 18S were optimal for developmental stage and low temperature, TUB and 18S showed the most stable expression for sex, and GAPDH and ACTIN were the best reference genes for monitoring gene expression at high temperature. Selection and validation of appropriate reference genes are critical steps in normalizing gene expression levels, which improve the accuracy and quality of expression data. Results of this study provide vital information on reference genes and is valuable in developing a standardized RT-qPCR protocol for functional genomics research in L. trifolii.

The polyphagous agromyzid fly, Liriomyza trifolii, is a significant and important insect pest of ornamental and vegetable crops worldwide. The adaptation of insects to different environments is facilitated by heat shock proteins (HSPs), which play an important role in acclimation to thermal stress. In this study, we cloned and characterized five HSP-encoding genes of L. trifolii (Lthsp20, Lthsp40, Lthsp60, Lthsp70, and Lthsp90) and monitored their expression under different thermal stresses using real-time quantitative PCR. Pupae of L. trifolii were exposed to 19 different temperatures ranging from -20 to 45°C. The results revealed that Lthsp20, Lthsp40, Lthsp70 and Lthsp90 were significantly upregulated in response to both heat and cold stress, while Lthsp60 was induced only by heat temperatures. The temperatures of the onset (Ton) and maximal (Tmax) expression of the five Lthsps were also determined and compared with published Ton and Tmax values of homologous genes in L. sativae and L. huidobrensis. Although L. trifolii occurs primarily in southern China, it has cold tolerance comparable with the other two Liriomyza species. Based on the heat shock proteins expression patterns, L. trifolii has the capacity to tolerate extreme temperatures and the potential to disseminate to northern regions of China.