Transcription factors, cap 'n' collar isoform C regulates the expression of CYP450 genes involving in insecticides susceptibility in Locusta migratoria.

BACKGROUND: The cap 'n' collar (Cnc) belongs to the Basic Leucine Zipper (bZIP) transcription factor super family. Cap 'n' collar isoform C (CncC) is highly conserved in the animal kingdom. CncC contributes to the regulation of growth, development, and aging and takes part in the maintenance of homeostasis and the defense against endogenous and environmental stress. Insect CncC participates in the regulation of various kinds of stress-responsive genes and is involved in the development of insecticide resistance. RESULTS: In this study, one full-length CncC sequence of Locusta migratoria was identified and characterized. Upon RNAi silencing of LmCncC, insecticide bioassays showed that LmCncC played an essential role in deltamethrin and imidacloprid susceptibility. To fully investigate the downstream genes regulated by LmCncC and further identify the LmCncC-regulated genes involved in deltamethrin and imidacloprid susceptibility, a comparative transcriptome was constructed. Thirty-five up-regulated genes and 73 down-regulated genes were screened from dsLmCncC-knockdown individuals. We selected 22 LmCncC-regulated genes and verified their gene expression levels using RT-qPCR. Finally, six LmCYP450 genes belonging to the CYP6 family were selected as candidate detoxification genes, and LmCYP6FD1 and LmCYP6FE1 were further validated as detoxification genes of insecticides via RNAi, insecticide bioassays, and metabolite identification. CONCLUSIONS: Our data suggest that the locust CncC gene is associated with deltamethrin and imidacloprid susceptibility via the regulation of LmCYP6FD1 and LmCYP6FE1, respectively.

DEAD-Box RNA Helicase DDX47 Maintains Midgut Homeostasis in Locusta migratoria.

Tissue homeostasis is critical for maintaining organ shape, size, and function. The condition is regulated by the balance between the generation of new cells and the loss of senescent cells, and it involves many factors and mechanisms. The midgut, an important part of the intestinal tract, is responsible for digestion and nutrient absorption in insects. LmDDX47, the ortholog of DEAD-box helicase 47 from Locusta migratoria, is indispensable for sustaining a normal midgut in the nymphs. However, the underlying cellular and molecular mechanisms remain to be elucidated. In this study, LmDDX47 knockdown resulted in atrophy of the midgut and gastric cecum in both nymph and adult locusts. After LmDDX47 knockdown, the number of regenerative and columnar cells in the midgut was significantly reduced, and cell death was induced in columnar tissue. LmDDX47 was localized to the nucleolus; this was consistent with the reduction in 18S rRNA synthesis in the LmDDX47 knockdown group. In addition, the acetylation and crotonylation levels of midgut proteins were significantly increased. Therefore, LmDDX47 could be a key regulator of midgut homeostasis, regulating 18S rRNA synthesis as well as protein acetylation and crotonylation in the migratory locust.

Characteristics of Halloween genes and RNA interference-mediated functional analysis of LmCYP307a2 in Locusta migratoria.

Halloween genes are involved in the biosynthesis of the molting hormone, which plays a key role in insect ecdysis, development, metamorphosis, and reproduction. Our previous work identified five Halloween genes from Locusta migratoria, but their functions are currently unknown. In this study, the sequences of these five Halloween genes were analyzed and characterized. LmCYP307a2, LmCYP306a1, LmCYP302a1, and LmCYP315a1 were primarily expressed in the prothoracic glands, while LmCYP314a1 was universally expressed in peripheral tissues, especially in the ovaries and Malpighian tubules. All five Halloween genes were mainly expressed from the 5th to the 7th d in 5th-instar nymphs. RNA interference (RNAi) silencing of LmCYP307a2 resulted in severe molting delays and molting failure, which could be rescued by supplementary 20-hydroxyecdysone. A hematoxylin and eosin staining analysis suggested that the RNAi of LmCYP307a2 inhibited the ecdysis process by inhibiting the apolysis and degradation of the old cuticle, and by promoting the synthesis of a new cuticle. Quantitative reverse transcription polymerase chain reaction results showed that the expressions of LmE74, LmCht5, and LmCht10 were dramatically down-regulated, while that of LmChsI was substantially up-regulated, after knockdown of LmCYP307a2. The results suggest that LmCYP307a2 is related to the molt process via regulation of chitin synthesis and degradation.

The microRNA miR-184 regulates the CYP303A1 transcript level to control molting of Locusta migratoria.

Cytochrome P450 monooxygenases (CYPs) play essential physiological functions in insects. CYP303A1 is highly conserved in insect species studied to date, and shows an indispensable role for adult eclosion in both Locusta migratoria and Drosophila melanogaster. However, how CYP303A1 is regulated to control insect developmental processes remains uninvestigated. In this study, we discovered functional binding sites for miR-184 in the coding sequence of LmCYP303A1. The luciferase reporter assay showed that miR-184 could target LmCYP303A1 and regulate its expression in vitro. Furthermore, overexpression of miR-184 through microinjection of agomir to locusts reduced the transcripts of LmCYP303A1 and led to abnormal molting, which is similar to the phenotype of silencing LmCYP303A1 by direct injection of dsLmCYP303A1 to locusts. Meanwhile, down-regulation of miR-184 by injection of antagomir increased the LmCYP303A1 transcript and caused molting defects. These findings suggested that miR-184 could target LmCYP303A1 to regulate the molting process in L. migratoria, which might be considered as a novel target for pest control.

A dsRNA-degrading nuclease (dsRNase2) limits RNAi efficiency in the Asian corn borer (Ostrinia furnacalis).

The efficiency of RNA interference (RNAi) varies substantially among different insect species. Rapid degradation of double-stranded RNA (dsRNA) by dsRNA-degrading nucleases (dsRNases) has been implicated to cause low RNAi efficiency in several insect species. In this study, we identified four dsRNase genes (OfdsRNase1, OfdsRNase2, OfdsRNase3 and OfdsRNase4) from the Asian corn borer (Ostrinia furnacalis) transcriptome database. Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides. Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae. RNAi efficiency was investigated in 2-d-old fifth-instar larvae (high expression of dsRNase2) and 2-d-old pupae (low expression of dsRNase2) by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein (OfLgl). Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae, but not in larvae, suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages. This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene, OfHex1, was significantly improved after knockdown of OfdsRNase2. When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro, only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA. Taken together, our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O. furnacalis larvae.

Identification of LmUAP1 as a 20-hydroxyecdysone response gene in the chitin biosynthesis pathway from the migratory locust, Locusta migratoria.

In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N-acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene LmCHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20-hydroxyecdysone (20E) is still largely unclear. Here, we showed the developmental expression pattern of LmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference (RNAi) directed toward a common region of the two isoforms of LmEcR (LmEcRcom) reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection-based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late-response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi-based pest control.

Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria.

Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development.

RNA interference to reveal roles of ß-N-acetylglucosaminidase gene during molting process in Locusta migratoria.

ß-N-acetylglucosaminidases are crucial enzymes involved in chitin degradation in insects. We identified a ß-N-acetylglucosaminidase gene (LmNAG1) from Locusta migratoria. The full-length complementary DNA (cDNA) of LmNAG1 consists of 2 667 nucleotides, including an open reading frame (ORF) of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5'- and 3'-ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized ß-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real-time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA (dsRNA). As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAG1-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process of L. migratoria.

Bacterial and fungal diversity in the starter production process of Fen liquor, a traditional Chinese liquor.

Fermented foods and beverages are important parts of human diet. Fen liquor, a Chinese liquor is a fermented beverage that uses a traditional fermentation process. Starters are the main microbial source and also provide nutrients for microorganisms during fermentation. In this study, starters of Fen liquor were produced through a complex traditional fermentation process. To investigate the community structure and the composition of microorganisms in the starter production process, bacterial 16S rRNA and fungal internal transcribed spacer (ITS) regions were sequenced using clone libraries and pyrosequencing, respectively. There was much higher diversity among the bacteria than among the fungi in the starter production process. Bacteria on the surface of the starters belonged mostly to the Lactobacillaceae family, while members of the Bacillacae family were dominant in the interior of the samples that lacked access to air and water. In the fungi population, diversity was high only in the raw material. In all other samples, nearly all of the fungal sequences were from Pichia kudriavzevii, a member of the Saccharomycetaceae family. Nearly all samples showed similar fungal community structures, indicating that there was little change in the fungal community. To the best of our knowledge, this is the first report to reveal the whole process of the starter production of Chinese traditional liquor. The findings obtained in this study provide new insights into understanding the composition of the microbial community during the traditional Chinese liquor starter production process and information about the production process control and monitoring.

The complete mitochondrial genome of Sasakia funebris (Leech) (Lepidoptera: Nymphalidae) and comparison with other Apaturinae insects.

Sasakia funebris, a member of the lepidopteran family, Nymphalidae (superfamily Papilionoidea) is a rare species and is found only in some areas of South China. In this study, the 15,233 bp long complete mitochondrial genome of S. funebris was determined, and harbors the gene arrangement identical to all other sequenced lepidopteran insects. The nucleotide composition of the genome is highly A+T biased, accounting for 81.2%. All protein-coding genes (PCGs) start with typical ATN codons, except for COI which begins with the CGA codon. All tRNAs have a typical clover-leaf secondary structure, except for tRNASer(AGN), the dihydrouridine (DHU) arm of which forms a simple loop. The S. funebris A+T-rich region of 370 bp contains several features common to the Lepidoptera insects, including the motif ATAGA followed by a 19 bp poly-T stretch, and two tandem repeats consisting of 18 bp repeat units and 14 bp repeat units. The phylogenetic analyses of Apaturinae based on mitogenome sequences showed: (S. funebris+Sasakia charonda)+(Apatura metis+Apatura ilia). This result is consistent with the morphological classification.

Bacterial and fungal diversity in the traditional Chinese liquor fermentation process.

This study endeavored to investigate the variability of bacteria and fungi present during the fermentation process of the light-fragranced distilled liquor known as Fen liquor. To accomplish this, we used a combination of clone libraries of 16S rRNA genes, bar-coded pyrosequencing of the internal transcribed spacer region 1 (ITS1), and quantitative real-time PCR (qPCR). Fifteen families of bacteria and six families of fungi were detected. More than 91% of 16S rRNA gene sequences could be assigned to the family Lactobacillaceae, which were then classified to eight different operational taxonomic units (OTUs), based on a 3% cut-off. The most abundant OTU which contributed to 51% of the total 16S rRNA gene sequences was affiliated with Lactobacillus acetotolerans and had a significantly similar variation trend with the chemical constituents detected. Sixty percent of the fungal ITS1 region sequences were affiliated with the family Saccharomycetaceae. The most abundant OTU was very similar to Issatchenkia orientalis, which displayed notable similarities with respect to the change trends in both ethanol and organic acid contents. The sequences of the second most abundant OTU were closest to Saccharomyces cerevisiae, an important species in the process of ethanol production. Furthermore, about one fourth of the ITS1 region sequences belonged to the family Saccharomycopsidaceae. Conversely, very few sequences could be grouped together with filamentous fungi. The results of qPCR showed that the content of bacteria was increased while that of fungi was more stable in the fermentation process. It is very important to simultaneously investigate bacterial and fungal variations in food-fermentation processes.

[Genetic diversity of different geographical populations of Oxya chinensis based on AFLP analysis].

In order to study the genetic diversity and genetic structure of populations, 7 populations of Oxya chinensis from 7 provinces (or cities) of China were analyzed using AFLP technique. A total of 336 reproducible bands were amplified with 7 primer combinations from 128 individuals. Two hundred and ninety-two bands (86.90%) were polymorphic. High genetic diversity was found among O. chinensis populations and Wanning population had higher genetic diversity than other populations. Mantel test (r=0.27, P=0.89) suggested that there was no significant association between genetic distance and geographic distance. Remarkable genetic differentiation was found among populations. Unweighted pair group method average (UPGMA) tree showed that the 7 O. chinensis populations were divided into 3 groups: Changping of Beijing, Tai-yuan of Shanxi and Jining of Shandong populations in the north; Hanzhong of Shaanxi, Changsha of Hunan and Laibin of Guangxi populations in the south; and Wanning of Hainan population. Principal component analysis indicated significant genetic differentiation of the north and the south populations and island and continent populations existed in the 7 O. chinensis populations because of geographic isolation.

Oxidative stress related enzymes in response to chromium (VI) toxicity in Oxya chinensis (Orthoptera: Acridoidae).

The toxic effects of Cr(VI) on antioxidant enzymes of Oxya chinensis (Orthoptera: Acridoidae) were determined. Changes in the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPx) were measured in O. chinensis insects injected with Cr(VI). Fifth-nymphs of O. chinensis insects were injected with Cr(VI) with different concentrations (0, 75, 150, 225, 300, 375, 450 mg/kg of body weight). The results showed that Cr(VI) led to the change of SOD, CAT, and GPx activities at different concentrations, which revealed that: (1) The oxidative stress of SOD increased with the increase of Cr(VI) concentration. (2) With the increase of Cr(VI) concentrations, CAT activities for females increased at lower concentrations, but decreased at higher concentration range, which indicated that antioxidant system of O. chinensis was not influenced by the presence of Cr(VI). A very similar response to Cr(VI) effect for males indicated that Cr(VI) concentrations were not high enough to damage O. chinensis in terms of CAT. (3) The GPx activity for females increased in all treatments, which revealed that the damage power of Cr(VI) was increased with the increase of Cr(VI) concentrations in terms of GPx, but the effect was not so remarkable. There was not a consistent trend of GPx activities for males in all treatments of Cr(VI). Cr (VI)-induced changes in antioxidant enzymes were different for SOD, CAT and GPx, of which the tendency was that activities generally changed with increase of concentrations of Cr(VI) suggesting SOD, CAT, and GPx could serve as indices of oxidative stress to some extent.

Comparative allozyme analysis of two geographic populations of Pararcyptera microptera meridionalis in China.

The genetic structure of the two populations of Pararcyptera microptera meridionalis (Ikonn.) from Hebei and Liaoning in China was analyzed with horizontal starch gel electrophoresis. Among 15 loci of 11 enzymes identified in zymograms, Adk-1, Fbp-1, Mdh-2 and G3pd-1 showed low variability with few alleles. Higher allelic polymorphisms were observed at Fbp-2, Mdh-1 and Me-1. The two populations demonstrated high percentage of polymorphic loci (93.3% and 100.0%) but low observable overall heterozygosity (0.061 and 0.086), that could be attributed to heterozygote deficiencies, which led to the genotype frequency deviating from Hardy-Weinberg expectations. It is reasoned that the strong movement capability of the insect makes the individuals likely to be exposed to drastically varied environments, which tends to maintain dynamic equilibrium of genetic polymorphisms. The F-statistics between the two populations was comparatively smaller ( F(st) = 0.084), but larger when compared with those in migratory locusts like Locusta migratoria manilensis. Nei's genetic identity (I) and Roger's genetic distance (D) also showed close genetic relationship of the two populations by their high genetic identity (I = 0.904) and small genetic distance (D = 0.256). However,considerably qualitative and quantitative differences were noted at loci Acp-1 (F(st) = 0.462) and Pgi-1 (F(st) = 0.182).

[Genetic relationships among five populations of Oxya chinensis in Shanxi Province and adjacent region based on RAPD].

Random Amplified Polymorphic DNA (RAPD) markers were applied to analyze genetic relationships of five populations of Oxya chinensis collected from Shanxi Province and laner Mongolia, Oxya japonica from Guangxi was used as an outgroup. Genomic DNA of sixty-four individuals was extracted from dissected leg muscle using phenol-chloroform procedure, and then amplified by 10 random primers (10 bp), the amplified products were separated by agarose gel electrophoresis. The results were as follows: (1) a total of 115 clear and reproducible bands were generated, molecular size was 200 - 2500 bp. The obtaining segments of individual primer were among 5 - 15, on average, about 12 bands per primer. (2) The dendrogram based on 115 RAPD markers was constructed and clustered using between-groups linkage method. The cluster analysis indicated strong similarities within populations, firstly, the individuals in each population closely clustered together;and then five populations of Oxya chinensis could be distinguished with RAPD markers and were grouped into two distinct clusters. The dendrogram showed that Shanxi Linyi population and Tunliu population were the most similar,which were clustered with Taiyuan population Shanxi into one cluster, while, Daixian population in Shanxi was closely related to laner Mongolia population, both of which belonged to the other cluster. Nevertheless, All the five populations of Oxya chinensis had far genetic distance with Oxya japonica. In the dendrogram, a tendency of clustering following a North-South gradient could be observed, the results implied that genetic distance of five populations of Oxya chinensis correlated with geographical distance to some degree.

[Differential acute mortality among the allozyme genotypes of Oxya chinensis by pesticide avermectin].

The rice grasshopper Oxya chinensis exhibits polymorphic loci at Ldh, Gpi, Pgm and Me. The data of the mean number of alleles per locus (A = 2.8), percentage of polymorphic loci (P = 80.0%), the observed mean heterozygosities (Ho = 0.271 approximately 0.279) and the expected mean heterozygosities (He = 0.305 approximately 0.316) of the species suggest that O. chinensis possesses sufficient genetic diversity. It was hypothesized that the high polymorphisms at Ldh, Gpi, Pgm and Me might make it possible for pesticide avermectin to act as a selective agent through differential lethality among the insect individuals with different genotypes. In this study a total of 855 grasshoppers were injected with avermectin (1.3 x 10(-2) g/g) to obtain a mortality of 54% after 24 hours. The allozyme analysis was then employed to determine the genotypes of Ldh, Gpi, Pgm and Me for both dead and surviving individuals. Contingency table chi2 tests showed that avermectin displayed random lethal effects on the genotypes at the loci of Ldh, Pgm and Me, without correlation between the genotype and mortality. In contrast, at Gpi locus, the grasshopper demonstrated a mortality cline of Gpi-AA (38%), Gpi-AB (51%), Gpi-BB (58%) and Gpi-BC (74%). The significant mortality differences were found among the following genotype pairs: Gpi-AA vs. Gpi-BB, Gpi-AA vs. Gpi-BC and Gpi-AB vs. Gpi-BC. These data implied the Gpi-AA genotype was likely related to the specie's resistance to the pesticide avermectin. It was also noted that the Gpi-A allele was present in the genotypes with low morality,while Gpi-B was present in the genotypes with moderate mortality, and the individuals with Gpi-C allele exhibited the highest mortality. The data obtained in this study suggested that the increasing proportion of Gpi-AA genotype and perhaps Gpi-A allele in a population may be useful as a potential resistant biomarker of O. chinensis to pesticide avermectin.

[Impacts of malathion on population genetic structure of Oxya chinensis].

Allozyme electrophoresis was employed to compare the difference in mortality among the genotypes at two polymorphic loci of Pgm and Me of grasshopper Oxya chinensis individuals acutely exposed to 1.5g/L malathion which resulted in 56% mortality in 24 hours. The selective lethal effects were observed among the genotypes at Pgm locus but not at Me locus. It is noted that the genotype Pgm-ab experienced the highest mortality (80%), whereas Pgm-bb and Pgm-bc were 49%, lower than the average. The chi(2) tests showed significant difference in morality between Pgm-bb and Pgm-cc. After exposure the allele frequency of Pgm-b showed a notable increase among surviving individuals. The cluster analysis based on Roger's genetic distance indicated that the acute exposure to malathion can cause differentiation in genetic composition at population level in Oxya chinensis. Because malathion is commonly used as the insecticide for grasshopper control, the data obtained in this study suggest that the similar genotype-mortality effects may occur in crop fields.

[Molecular phylogenetic relationships among species in Oxya serville(Orthoptera: Catantopidae) based on random amplified polymorphic DNA(RAPD)].

The molecular phylogenetic relationships of five species of Oxya Audient-serville including O. chinensis, O. brachyptera, O. agavisa, O. japonica and O. intricata were studied with RAPD. Genomic DNA of forty-one individuals were amplified with eight oligonucleotide (10 mer) primers which were previously selected, the specifical bands occured in all them, a total of 96 clear and reproducible bands (rang from 200-2500 bp) were generated, 95 of which were polymorphic band, the only one band (850 bp) which was amplified with S362 was common to five species of Oxya. The obtaining segments of individual primer were between 8-16, the average was 12. A molecular phylogenetic tree based on was constructed Euclidean distance among five species of rice grasshopper by between-groups linkage method, the result indicated O. chinensis was closely related to O. brachyptera, the genetic relationship of O. japonica and O. agavisa was also close, whereas O. intricata had far phylogenetic relationship with them. The results of dendrogram were consistent with the previous conclusion of morphologic classification and cytotaxonomy, and suggested RAPD was suitable for analysis of phylogenetic relationships among species of Oxya.

Genetic differentiation of different populations of four locust species in China.

The genetic structure of eight populations of four locust species in three families (Catantopidae, Pamphagidae and Pyrgomorphidae) was examined by means of horizontal starch gel electrophoresis, the locusts were collected from Shanxi, Jiangsu and Hebei Province in China. The allele frequency and allozyme polymorphism of 12 enzymes (18 loci) were analyzed. Low variability with a few alleles was observed in Shirakiacris shirakii and Atractomorpha sinensis, however, Oxya Chinensis and Haplotropis brunneriana showed high polymorphism. In the eight populations of the four locust species, high percentage of polymorphic loci was observed (53.3%-100.0%). Owing to heterozygote deficiency of some species, all the four species demonstrated low overall heterozygosity (Ho = 0.034-0.139), which lead to the genotype frequency deviated significantly from Hardy-Weinberg equilibrium. However, many of the examined loci were fit for or close to H-W equilibrium in Atractomorpha sinensis. A certain differentiation in mean heterozygosity was found among the four species. Due to the difference of migratory capability, reproductive manner and living bound, mean heterozygosity (Ho) is higher in Haplotropis brunneriana (Ho = 0.089-0.139) and Oxya Chinensis (Ho = 0.073-0.090) than in Atractomorpha sinensis (Ho = 0.034-0.050) and Shirakiacris shirakii (Ho = 0.048-0.068). Genetic differentiation from F-statistics was also different at population level among four locust species. It was high in Haplotropis brunneriana (Fst = 0.32) and Atractomorpha sinensis (Fst = 0.31), and low genetic differentiations were observed in Oxya Chinensis (Fst = 0.20) and Shirakiacris shirakii (Fst = 0.18). It was confirmed that migratory capability, adaptability and surrounding factors had an important influence on the genetic differentiation of locust populations. The taxon relationships based on Nei's genetic identity (I) and genetic distance (D) were consistent with results obtained from karyotypic analyses. Oxya Chinensis and Shirakiacris shirakii in the same family have a higher genetic identity (I = 0.576) and a smaller genetic distance (D = 0.559); the species examined in Pampagidae displayed somewhat closer relationship to those in Pyrgomorphidae (D = 0.776, I = 0.464) than to those in Catantopidae (D = 0.908, I = 0.406). It is suggested that the allozyme analysis is an useful molecular marker for phylogenetic reconstruction of locust at both species and population levels.

Genetic studies on eight populations of eight locust species from Shanxi Province, China.

The genetic structure of eight locust species in three families (Catantopidae, Oedipodidae and Arcypteridae) from Shanxi Province in China was compared using allozyme analysis with horizontal starch gel electrophoresis. Among 17 loci identified in zymograms, Ao-1, Est-3, G3pd-1, Idh-2 and Mdh-2 had low variability with a few alleles. High polymorphism was observed at Ldh-1, Me-1 and Gpi-1. Each of the eight species demonstrated high percentage of polymorphic loci (P = 64.7%-94.1%) but low observed heterozygosity (H0 = 0.024-0.087) due to heterozygote deficiency. It was noted that the migratory locusts usually had higher percentage of polymorphic loci (P = 88.2%-94.1%) than non-migratory species (P = 64.7%-94.1%). The only exception is Oxya chinensis(P = 94.1%). It is reasoned that the higher polymorphism is necessary for migratory species to cope with the environments that might be drastically different from the habitats before migration. The taxon relationships using cluster analysis based on Nei's genetic identity (I) and Roger's genetic distance (D) were the same at species and genus levels. The differences were found at family level, possibly due to the alternative algorithms. The cladogram using Roger's genetic distance (D) overlapped the relationship obtained from karyotypic analyses, which demonstrated that the species examined in Catantopidae displayed somewhat closer relationship to those in Oedipodidae than to those in Arcypteridae. It is suggested that the allozyme analysis is useful as molecular marker for locusts in phylogenetic reconstruction at the species and genus level, while additional data from other studies are necessary when used for higher taxa.