Development and parentage analysis of SNP markers for Chestnut-vented Nuthatch(Sitta nagaensis)based on ddRAD-seq data

Extra-pair paternity(EPP)is commonly found in socially monogamous birds,especially in small passerine birds,and there are interspecific and intraspecific variations in the extent of EPP.The Chestnut-vented Nuthatch(Sitta nagaensis)is a socially monogamous passerine bird,and verifying whether this species has EPP relies on parentage testing-S.nagaensis is not known to have EPP.In this study,we developed SNP markers of this species that are informative for parentage analysis from double digest restriction site-associated DNA sequencing(ddRAD-seq)data.A panel consisting of 50 SNP markers,with a mean heterozygosity of 0.343,was used to resolve 95% of nestlings to fathers.The combined exclusion probabilities for the first parent and second parent were 0.991 and 0.9999,respectively.This panel of SNP markers is a powerful tool for parentage assignments in S.nagaensis.In addition,we found that three offspring(7.9%)from three nests(23.1%)were the result of extra-pair fertilization out of 38 offspring in 13 nests.Our study provided information on parentage analysis that has not been reported before in S.nagaensis.It also supplemented the understudied EPP behavior of birds in Asia,contributing to a general understanding of the EPP behaviors of birds.

Leaf choice in black-and-white snub-nosed monkeys Rhinopithecus bieti is related to the physical and chemical properties of leaves

To understand the effects of the chemical and physical properties of plant leaves on food choice in Rhinopithecus bieti, we collected mature leaves of nine food and five non-food plant species at the southernmost part of the species＇s range. Chemical properties such as fat, ash, crude protein （CP）, total phenolics （TP）, and fiber content including neutral detergent fiber （NDF）, acid detergent fiber （ADF）, lignin, celluloses and hemicelluloses, and physical toughness were measured. R. bieti tended to choose leaves with lower fiber content, higher ash, a higher ratio of CP/ADF, and lower toughness. No difference was found for fat, crude protein, total phenolics, hemicelluloses and lignin between food and non-food leaves. Even though the ratio of CP/ADF is generally regarded as a good indicator for colobine food choice, the difference in the ratio of CP/ADF between food and non-food leaves was only the result of differences in ADE Since positive correlations were found between ADF and tough- ness from all leaves （both food and non-food species）, and toughness of leaves was likely easier for R. bieti to sense than fiber content via mastication, the toughness of leaves may function as a predictor of food choice in this species [Current Zoology 56 （6） 643-49, 2010].

Improvement and Exploration of Teaching Methods for General Entomology in Agriculture and Forestry Colleges and Universities

According to characteristics of General Entomology and existing problems in teaching process,it came up with methods and countermeasures for improving teaching General Entomology,including improving practical ability of students through enhancing all links of practice teaching,and stimulating learning interest of students through improving methods of course examination.

Effects of tsaoko (Fructus tsaoko) cultivating on tree diversity and canopy structure in the habitats of eastern hoolock gibbon (Hoolock leuconedys)

In this study, the quadrat method was used to study the effects of tsaoko （Fructus tsaoko） plantation on tree diversity and canopy structure of two natural habitats of eastern hoolock gibbon （Hoolock leuconedys）： Nankang （characterized by extensive tsaoko plantation） and Banchang （relatively well reserved and without tsaoko plantation）. Totally, 102 tree species from 25 families and 16 woody liana species from 10 families were recorded in Nankang, whereas 108 tree species from 30 families and 17 woody liana species from 12 families were recorded in Banchang. Although the tree species between two habitats is different, both habitats are characterized by enriched food resources for eastern hoolock gibbons, sharing similar dominant plant families. Due to tsaoko plantation, tree density proportion and diversity of forest layer I （〉20 m） in Nankang were both significantly decreased, but the tree density of layerH （10-20 m） increased. Likewise, in conjunction with these behavioral observations, we also address potential impacts of tsaoko plantation on the behavior of eastern hoolock gibbon.

Occurrence of internally ovipositing non-agaonid wasps and pollination mode of the associated agaonid wasps

Figs(Ficus, Moraceae) and their pollinating fig wasps(Hymenoptera, Agaonidae, Chalcidoidea) are a striking example of obligate mutualism and coevolution. Agaonid females enter the figs to lay their eggs,but also actively or passively transport pollen into the figs at the same time. We examined eight related fig tree species pollinated by host specific Eupristina agaonids to determine the relationships between pollination mode, host pollen, ovule ratios and the ability of the figs to recruit additional non-agaonid pollinators. Uniquely amongst the eight Eupristina species, the pollinator of Ficus curtipes has nonfunctional pollen pockets and no coxal combs, showing that it pollinates passively. Reflecting this, the anther-to-ovule ratio of F. curtipes is unusually high.In addition to the agaonids, figs support many species of 'non-pollinating fig wasps'(NPFW) that are typically ovule gallers or parasitoids. These mainly oviposit from outside the figs but there are a few species of NPFW that are like agaonids and enter the figs to oviposit. Two of the eight Eupristina pollinated fig trees support host specific internally-ovipositing fig wasps belonging to the chalcidoid genera Diaziella(Sycoecinae) and Lipothymus(Otitesellinae). Reflecting the trees' pollination modes, these fig wasps act as supplementary pollinators of F. curtipes, but not of Ficus glaberrima, where agaonid pollination is active.

Genetic diversity and population genetic structure of Python bivittatus in China

The Burmese python （Python bivittatus） has recently suffered large population declines in the wild in China due to illegal capture, overexploitation, deforestation and the loss of its natural habitat. Greater knowledge of the genetic diversity and structure of wild P. bivittatus populations is needed to help ensure its effective management. In this study, we investigated the genetic diversity and population genetic structure of wild P. bivittatus in China in detail. 109 P. bivittatus individuals from five distribution areas in Guangdong （GD）, Guangxi （GX）, Hainan （HN）, Fujian （FJ） and Yunnan （YN） province of China were collected, and their genetic structure and diversity were analyzed. Eight highly polymorphic microsatellite loci were utilized to reveal high levels of genetic diversity in the P. bivittatus population. Genetic diversity was highest in GX, and lowest in GD. All geographic populations demonstrated a bottleneck effect indicating recent population decline. Fst and AMOVA analyses revealed that there was moderate genetic differentiation among the five populations, and that only 10.59 % of total genetic diversity occurred among populations. Fst values between pop- ulations were positively correlated with their geographical distances. Genetic structure analyses revealed that the HN, GX and GD populations, which were geographically closest, were assigned to a genetic cluster, while the YN and FJ populations constituted a single cluster, respectively.

Birth seasonality and pattern in black-and-white snub-nosed monkeys （Rhinopithecus bieti） at Mt. Lasha, Yunnan

Seasonal variation in environmental factors is vital to the regulation of seasonal reproduction in primates. Consequently, long-term systematic data is necessary to clarify the birth seasonality and pattern of primates in highly seasonal environments. This study indicated that black-and-white snub-nosed monkeys （Rhinopithecus bieti） at Mt. Lasha exhibited strict birth seasonality with a pulse model. Infants were born with a certain degree of synchronization. Birth distribution showed three birth peaks, and the birth pattern showed a ＂V＂ style in even-numbered years and a gradual increase in odd-numbered years. The beginning date, end date and median birth date were earlier in even-numbered years than those in odd-numbered years. The higher latitude of their habitats, earlier birth date, shorter birth period, fewer birth peaks and stronger birth synchrony might be adaptations for strongly seasonal variation in climate and food resources. After the summer solstice when daylight length began to gradually shorten, R. bieti at Mt. Lasha started to breed during the period with the highest environmental temperature and food availability, which implied that photoperiod may be the proximate factor triggering the onset of estrus and mating. It appears that R. bieti coincided conception and mid-lactation with the peak in staple foods, and weaning with the peak in high quality of foods. Thus, food availability was the ultimate factor regulating reproductive seasonality, and photoperiod was the proximate factor fine-turning the coordination between seasonal breeding and food availability.

Analysis of Genetic Differences among Monokaryon Strains of Flammulina velutipes Using SCoT and ISSR Markers

[Objective]This study aimed to analyze genetic differences among Flammulina velutipes monokaryon strains.[Method]Twenty F1monokaryon strains（W1-W20）of F.velutipes were separated with conventional dilution method.Fifteen SCoT primers and nine ISSR primers were screened.According to amplification results,genetic similarity coefficients among various strains were calculated using NTsys 2.10e analysis software for cluster analysis.[Result]Six primers could be use to amplify clear polymorphic bands.To be specific,a total of 327 clear DNA fragments were amplified,including 287 polymorphic bands,accounting for 87.77%of the total number of bands amplified.Based on SCoT analysis,the genetic identity（GI）among 20 strains ranged from 0.187 5 to 0.937 5;to be specific,GI between W2 and W3 and that between W11 and W12 reached the maximum of 0.9375;GI between W15 and W18 was the minimum of 0.187 5.Based on ISSR analysis,GI among 20 strains ranged from 0.250 0 to 1.000 0;to be specific,GI between W3,W4 and W9,GI between W15 and W17,and that between W16 and W19 reached the maximum of 1.000;GI between W14 and W18 was the minimum of 0.250 0.Such low genetic identity fully demonstrated great genetic differences among F.velutipes monokaryons.According to results of cluster analysis,at a similarity level of 0.55,20 F.velutipes monokaryons were significantly divided into three groups using SCoT markers;at a similarity level of 0.66,20 F.velutipes monokaryons were divided into three groups using ISSR markers.Specifically,W11,W18 and W20 were invariably divided into one group;W15 and W17 were divided into one subgroup.[Conclusion]In this study,two type of markers were used for analysis of genetic diversity among F.velutipes monokaryon strains,which provided scientific and practical basis for screening high-quality monokaryon parents of F.velutipes.

Potential Geographic Distribution of Oriental Fruit Fly in Jiangxi Province

[Objective] The aim was to effectively monitor and manage oriental fruit fly.[Method] Maxent model and ArcGIS were used to predict potential geographic distributions of the oriental fruit fly based on associations between known occurrence records and a set of environmental variables.[Result] The suitable areas for Bactrocera dorsalis infestations were mainly restricted to central and southern Jiangxi Province,with Latitude ranged from 24 to 28°N.Northeast Jiangxi,Northwest Jiangxi and the regions bording upon Yangtse River were predicted as unsuitable for Bactrocera dorsalis.The fit for the model as measured by AUC was high,with value of 0.978 for the training data and 0.965 for the test data,indicating the high level of discriminatory power for the Maxent.A jackknife test in Maxent indicated that mean temperature of coldest quarter with highest gain value was the most important environmental variable that restricted the expansion to north Jiangxi Province.[Conclusion] Further research into the biology of the species and their ability to overcome barriers was necessary to explain niche differentiation and better understand invasion risk.

Prediction of Potential Distribution of Quadrastichus erythrinae Kim in China

Based on Maxent niche model and combined with ArcGIS,the suitable area range for Quadrastichus erythrinae Kim in China was predicted in the paper.The results showed that high suitable area for Q. erythrinae in China included most northeast coastal areas of Hainan Island,partial southern coastal area of Guangdong Province,partial northwestern coastal area and partial southeast coastal area of Taiwan Island; moderate suitable area included partial area of Hainan,some contiguous areas of Guangxi and Guangdong,most areas of Guangdong,partial area of Fujian and Taiwan; low suitable area included partial area from northwestern coast to inland of Hainan Island,west coastal area of Taiwan Island,most area in Guangxi,partial areas in Guangdong,Fujian and Yunnan.

Olfactory responses of Aphidius gifuensis to odors of host plants and aphid-plant complexes

The olfactory responses ofAphidius gifuensis to odors from two host plants （Nicotiana tabacum and Brassica napus ssp.） and their complexes with different infestation levels of two host aphids （Myzuspersicae and Lipaphis erysimi） were respectively examined in an olfactometer. The results showed that female A. gifuensis did not respond to odors of undamaged or mechanically damaged host plants, but significantly responded to odors of aphid/plant complexes. Moreover, A. gifuensis responded significantly to odors of both M. persicae and L. erysimi/plant complexes when host plants were infested by high levels of aphids, suggesting that quantity of aphid-induced volatiles could be important for attracting A. gifuensis. When tested between aphid/plant complexes, .4. gifuensis did not show its preference for either complex. The efficiency of.4. gifuensis against aphids in open fields potentially could be improved by using its olfactory response to aphid/plant complexes.

Alternaria toxin-induced resistance against rose aphids and olfactory response of aphids to toxin-induced volatiles of rose plants

The search for active toxins for managing weeds or plant diseases is believed to be a promising avenue of investigation. However, the effects of Alternaria toxins on insects have just begun to be investigated. Bioactivities of toxins from four strains of Altemaria alternata on Rosa chinensis and rose aphid Macrosiphum rosivorum were tested in the present study. At a concentration of 50.0 pg/ml, the crude extract （toxin） of strain 7484 was found not to be harmful to rose plants with excised leaf-puncture method （P≥0.079）, and rose plants showed enhanced resistance to rose aphids when this Altemaria toxin was sprayed on the plants （P≤0.001）. However, this toxin caused no detrimental effects on aphids in insecticidal bioassay at a concentration of 10.0 to 160.0 μg/ml （P≥0.096）. Therefore, the Alternaria toxin had significantly induced the resistance of rose plants against rose aphids, demonstrating that the resistance mechanism triggered by the Altemaria toxin in the rose plant may also be used by the plant to defend itself against insects. Further bioassays aimed to discover the olfactory responses of aphids to the toxin-induced volatiles of host plants. The aphids were significantly more attracted to both volatiles emitted and collected from control rose plants than to both volatiles emitted and collected from the toxin-treated rose plants （P≤0.014）. This result showed that the toxin-induced resistance related to the volatile changes of host plants.

The diversity of soil culturable fungi in the three alpine shrub grasslands of Eastern Qilian Mountains

To understand the diversity of culturable fungi in soil at alpine sites, Rhododendron fruticosa shrubland, Salix cupularis fruticosa shrubland, and Dasiphoru fruticosa shrubland of the Eastern Qilian Mountains were selected to investigate. Three methods, including tradi- tional culturing, rDNA internal transcribed spacer （ITS） sequence analysis, and economical efficiency analysis, were carried out to estimate the diversity of soil culturable fungi of these three alpine shmblands. A total of 35 strains of culturable fungi were cultured by dilution plate technique and were analyzed by rDNA ITS sequence. The diversity indices such as species abundance （S）, Shannon-Wiener index （H）, Simpson dominance index （D）, and Pielou evenness index （3） of Rhododendron fruticosa shrubland, Salix cupularis fruticosa shrubland, and Dasiphoru fruticosa shrubland were ranged between 16 and 17, 2.66-2.71, 0.92, 0.95~）.97 respectively. The results showed that the diversity of soil fungi were abundant in these three types of alpine shrub grasslands, while further study should be done to explore their potential value.

Prophenoloxidase from Pieris rapae:gene cloning,activity,and transcription in response to venom/calyx fluid from the endoparasitoid wasp Cotesia glomerata

Prophenoloxidase (PPO) plays an important role in melanization, necessary for defense against intruding parasitoids. Parasitoids have evolved to inject maternal virulence factors into the host hemocoel to suppress hemolymph melanization for the successful development of their progeny. In this study, the full-length complementary DNA (cDNA) of a Pieris rapae PPO was cloned. Its cDNA contained a 2 076-base pair (bp) open reading frame (ORF) encoding 691 amino acids (aa). Two putative copper-binding sites, a proteolytic activation site, three conserved hemocyanin domains, and a thiol ester motif were found in the deduced amino acid sequence. According to both multiple alignment and phylogenetic analysis, P. rapae PPO gene cloned here is a member of the lepidopteran PPO-2 family. Injection of Cotesia glomerata venom or calyx fluid resulted in reduction of P. rapae hemolymph phenoloxidase activity, demonstrating the ability to inhibit the host's melanization. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of P. rapae PPO-2 in the haemocytes from larvae had not significantly changed following venom injection, suggesting that the regulation of PPO messenger RNA (mRNA) expression by venom was not employed by C. glomerata to cause failure of melanization in parasitized host. While decreased P. rapae PPO-2 gene expression was observed in the haemocytes after calyx fluid injection, no detectable transcriptional change was induced by parasitization, indicating that transcriptional down-regulation of PPO by calyx fluid might play a minor role involved in inhibiting the host's melanization.

Proteome changes in the plasma of Pieris rapae parasitized by the endoparasitoid wasp Pteromalus puparum

Parasitism by the endoparasitoid wasp Pteromalus puparum causes alterations in the plasma proteins of Pieris rapae. Analysis of plasma proteins using a proteomic approach showed that seven proteins were differentially expressed in the host pupae after 24-h parasitism. They were masquerade-like serine proteinase homolog (MSPH), enolase (Eno), bilin-binding protein (BBP), imaginal disc growth factor (IDGF), ornithine decarboxylase (ODC), cellular retinoic acid binding protein (CRABP), and one unknown function protein. The full length cDNA sequences of MSPH, Eno, and BBP were successfully cloned using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that the transcript levels of MSPH and BBP in the fat bodies of host pupae were inducible in response to the parasitism and their variations were consistent with translational changes of these genes after parasitism, while the transcript levels of Eno and IDGF were not affected by parasitism. This study will contribute to the better understanding of the molecular bases of parasitoid-induced host alterations associated with innate immune responses, detoxification, and energy metabolism.

Alternaria toxin-induced resistance in rose plants against rose aphid(Macrosiphum rosivorum): effect of tenuazonic acid

Many different types of toxins are produced by the fungus, Alternaria alternata （Fr.） Keissler. Little is known, however, regarding the influence of these toxins on insects. In this study, we investigated the toxin-induced inhibitory effects of the toxin produced by A. alternata on the rose aphid, Macrosiphum rosivorum, when the toxin was applied to leaves of the rose, Rosa chinensis. The results demonstrated that the purified crude toxin was non-harmful to rose plants and rose aphids, but had an intensive inhibitory effect on the multiplication of aphids. The inhibitory index against rose aphids reached 87.99% when rose plants were sprayed with the toxin solution at a low concentration. Further results from bioassays with aphids and high performance liquid chromatography （HPLC） analyses demon- strated that tenuazonic acid （TEA） was one of the most important resistance-related active components in the crude toxin. The content of TeA was 0.1199% in the crude toxin under the HPLC method. Similar to the crude toxin, the inhibitory index of pure TeA reached 83.60% 15 d after the rose plants were sprayed with pure TeA solution at the lower concentration of 0.060 IJg/ml, while the contents of residual TeA on the surface and in the inner portion of the rose plants were only 0.04 and 0.00 ng/g fresh weight of TeA-treated rose twigs, respectively, 7 d after the treatment. Our results show that TeA, an active component in the A. alternata toxin, can induce the indirect plant-mediated re- sponses in rose plants to intensively enhance the plant＇s resistances against rose aphids, and the results are very helpful to understand the plant-mediated interaction between fungi and insects on their shared host plants.

Heat shock protein genes （hsp20, hsp75 and hsp90） from Pieris rapae： Molecular cloning and transcription in response to parasitization by Pteromalus puparum

Most molecular work on the roles of heat shock proteins （hsps） in host-parasite interaction has focused on vertebrates, rather than invertebrates. Here the full length complementary DNA （cDNA） sequences of three hsp genes （hsp20, hsp75 and hsp90） were amplified from Pieris rapae, and their transcriptional responsiveness to parasitization by the endoparasitic wasp Pteromalus puparum were investigated. The cDNA sequence analysis of hsp20, hsp75 and hsp90 revealed open reading frames of 531, 2328 and 2 157 bp in length, which encode proteins with calculated molecular weights of 19.5, 75.48 and 82.7 kDa, respectively. The comparison of amino acid sequences showed that P rapae hsp20 shared highly divergent homology to that of other insects, while hsp75 and hsp90 showed high homology to their counterparts of other species. The expression analysis indicated that these three genes were influenced in response to parasitization by P. puparum. The hsp20 transcripts in parasitized pupae were higher compared to non- parasitized pupae. The expression of hsp75 and hsp90 were down-regulated following parasitization. The results indicate that hsps are involved in host-parasitoid interactions.

Fungal diversity notes 253-366:taxonomic and phylogenetic contributions to fungal taxa

Notes on 113 fungal taxa are compiled in this paper,including 11 new genera,89 new species,one new subspecies,three new combinations and seven reference specimens.Awide geographic and taxonomic range of fungal taxa are detailed.In the Ascomycota the new genera Angustospora(Testudinaceae),Camporesia(Xylariaceae),Clematidis,Crassiparies(Pleosporales genera incertae sedis),Farasanispora,Longiostiolum(Pleosporales genera incertae sedis),Multilocularia(Parabambusicolaceae),Neophaeocryptopus(Dothideaceae),Parameliola(Pleosporales genera incertae sedis),and Towyspora(Lentitheciaceae)are introduced.Newly introduced species are Angustospora nilensis,Aniptodera aquibella,Annulohypoxylon albidiscum,Astrocystis thailandica,Camporesia sambuci,Clematidis italica,Colletotrichum menispermi,C.quinquefoliae,Comoclathris pimpinellae,Crassiparies quadrisporus,Cytospora salicicola,Diatrype thailandica,Dothiorella rhamni,Durotheca macrostroma,Farasanispora avicenniae,Halorosellinia rhizophorae,Humicola koreana,Hypoxylon lilloi,Kirschsteiniothelia tectonae,Lindgomyces okinawaensis,Longiostiolum tectonae,Lophiostoma pseudoarmatisporum,Moelleriella phukhiaoensis,M.pongdueatensis,Mucoharknessia anthoxanthi,Multilocularia bambusae,Multiseptospora thysanolaenae,Neophaeocryptopus cytisi,Ocellularia arachchigei,O.ratnapurensis,Ochronectria thailandica,Ophiocordyceps karstii,Parameliola acaciae,P.dimocarpi,Parastagonospora cumpignensis,Pseudodidymosphaeria phlei,Polyplosphaeria thailandica,Pseudolachnella brevifusiformis,Psiloglonium macrosporum,Rhabdodiscus albodenticulatus,Rosellinia chiangmaiensis,Saccothecium rubi,Seimatosporium pseudocornii,S.pseudorosae,Sigarispora ononidis and Towyspora aestuari.New combinations are provided for Eutiarosporella dactylidis(sexual morph described and illus trated)and Pseudocamarosporium pini.Descriptions,illustrations and/or reference specimens are designated for Aposphaeria corallinolutea,Cryptovalsa ampelina,Dothiorella vidmadera,Ophiocordyceps formosana,Petrakia echinata,Phragmoporthe conformis and Pseudocamarosporium pini.The new species of Basidiomycota are Agaricus coccyginus,A.luteofibrillosus,Amanita atrobrunnea,A.digitosa,A.gleocystidiosa,A.pyriformis,A.strobilipes,Bondarzewia tibetica,Cortinarius albosericeus,C.badioflavidus,C.dentigratus,C.duboisensis,C.fragrantissimus,C.roseobasilis,C.vinaceobrunneus,C.vinaceogrisescens,C.wahkiacus,Cyanoboletus hymenoglutinosus,Fomitiporia atlantica,F.subtilissima,Ganoderma wuzhishanensis,Inonotus shoreicola,Lactifluus armeniacus,L.ramipilosus,Leccinum indoaurantiacum,Musumecia alpina,M.sardoa,Russula amethystina subp.tengii and R.wangii are introduced.Descriptions,illustrations,notes and/or reference specimens are designated for Clarkeinda trachodes,Dentocorticium ussuricum,Galzinia longibasidia,Lentinus stuppeus and Leptocorticium tenellum.The other new genera,species new combinations are Anaeromyces robustus,Neocallimastix californiae and Piromyces finnis from Neocallimastigomycota,Phytophthora estuarina,P.rhizophorae,Salispina,S.intermedia,S.lobata and S.spinosa from Oomycota,and Absidia stercoraria,Gongronella orasabula,Mortierella calciphila,Mucor caatinguensis,M.koreanus,M.merdicola and Rhizopus koreanus in Zygomycota.

Conserved profiles of digestion by double restriction endonucleases in insect genomes facilitate the design of dd RAD

Double-digested Restriction Site Associated DNA Sequencing（ddRAD） through next-generation sequencing（NGS） generates large numbers of loci for characterizing genomewide variation among multiple samples using next-generation sequencing. Different combinations of restriction endonucleases（REs） may produce varying size distributions of digested fragments, which affect the number of genotyped loci. Understanding digestion profiles across different species will help in selecting REs for digestion in a particular organism. In this study, we use of genome sequences to compare the in silico digestion profile of 26 combinations of REs in 131 insect species with two simulation programs. The number of digested fragments in the 300-450 bp range increases linearly with the size of the genome. Different species and insect orders showed similar profiles when digested by different combinations of REs in silico, indicating the conservation of digestion by double enzymes in insect genomes. Combinations with Nla III or TaqαI usually produced higher number of fragments in the range 300-450 bp, while combinations with EcoRI or MluCI produced fewer fragments. The proportion of fragments with the same overhangs at the two ends of digested DNA was higher than those with different overhangs. The two four-base enzyme pairs produced more fragments in the 300-450 bp range than pairs of four-base ＋ six-base enzymes. Experimental digestion of three species from Hymenoptera, Lepidoptera and Thysanoptera showed profiles congruent with in silico expectations. Our results shed light on understanding the digestion profiles of insect genomes and provide guidance on selecting REs for ddRAD projects.