The influence of first desaturase subfamily genes on fatty acid synthesis, desiccation tolerance and inter-caste nutrient transfer in the termite Coptotermes formosanus.

Desaturase enzymes play an essential role in the biosynthesis of unsaturated fatty acids (UFAs). In this study, we identified seven "first desaturase" subfamily genes (Cfor-desatA1, Cfor-desatA2-a, Cfor-desatA2-b, Cfor-desatB-a, Cfor-desatB-b, Cfor-desatD and Cfor-desatE) from the Formosan subterranean termite Coptotermes formosanus. These desaturases were highly expressed in the cuticle and fat body of C. formosanus. Inhibition of either the Cfor-desatA2-a or Cfor-desatA2-b gene resulted in a significant decrease in the contents of fatty acids (C16:0, C18:0, C18:1 and C18:2) in worker castes. Moreover, we observed that inhibition of most of desaturase genes identified in this study had a negative impact on the survival rate and desiccation tolerance of workers. Interestingly, when normal soldiers were reared together with dsCfor-desatA2-b-treated workers, they exhibited higher mortality, suggesting that desaturase had an impact on trophallaxis among C. formosanus castes. Our findings shed light on the novel roles of desaturase family genes in the eusocial termite C. formosanus.

An Acyl Carrier Protein Gene Affects Fatty Acid Synthesis and Growth of Hermetia illucens.

Acyl carrier protein (ACP) is an acyl carrier in fatty acid synthesis and is an important cofactor of fatty acid synthetase. Little is known about ACP in insects and how this protein may modulate the composition and storage of fatty acids. We used an RNAi-assisted strategy to study the potential function of ACP in Hermetia illucens (Diptera: Stratiomyidae). We identified a HiACP gene with a cDNA length of 501 bp and a classical conserved region of DSLD. This gene was highly expressed in the egg and late larval instars and was most abundant in the midgut and fat bodies of larvae. Injection of dsACP significantly inhibited the expression level of HiACP and further regulated the fatty acid synthesis in treated H. illucens larvae. The composition of saturated fatty acids was reduced, and the percentage of unsaturated fatty acids (UFAs) was increased. After interfering with HiACP, the cumulative mortality of H. illucens increased to 68.00% (p < 0.05). H. illucens growth was greatly influenced. The development duration increased to 5.5 days, the average final body weights of larvae and pupae were decreased by 44.85 mg and 14.59 mg, respectively, and the average body lengths of larvae and pupae were significantly shortened by 3.09 mm and 3.82 mm, respectively. The adult eclosion rate and the oviposition of adult females were also severely influenced. These results demonstrated that HiACP regulates fatty acid content and influences multiple biological processes of H. illucens.

Multiple Mating of Aphelinus asychis Enhance the Number of Female Progeny but Shorten the Longevity.

The Aphelinus asychis female adult is an important arrhenotocous parthenogenesis parasitoid of Myzus persicae, and its reproductive mode is beneficial for the population continuation of A. asychis by way of multiple mating and backcross. To explore the effect of mating on the population fitness and control efficiency of A. asychis, its mating frequency and backcross were observed under laboratory conditions. The results showed that most matings in A. asychis involved four distinct stages: courtship, pre-copulatory, copulation, and post-copulatory behaviours. Only the duration of courtship increased significantly with an increase in copulation frequency for females, and the courtship duration of A. asychis females mated with different males were significantly shorter than those mated with the same male at the same mating times, which suggested that A. asychis females might prefer to mate with different males to enrich the genotype of their offspring. The total number of mummified aphids and the female and male longevity decreased significantly with an increase in mating frequency. On the contrary, female progenies increased significantly with an increase of mating frequency, suggesting that sperm limitation might occur in females when they only mated once. These results imply that females might prefer to receive more sperm by mating multiple times in their life span. In addition, we found that the intrinsic rate of increase (r) of A. asychis of the control group (0.2858 d-1) was significantly greater than that in the backcross treatment (0.2687 d-1). The finite killing rate (θ) of A. asychis of the control group was similar to that in the backcross treatment, which showed that this treatment had a negligible negative effect on the control efficiency of A. asychis. In conclusion, the results showed that multiple mating increased the number and proportion of A. asychis female progenies but shortened the longevity of female and male adults, while the negative effect of backcross on the control efficiency of A. asychis was negligible.

[Cross-resistance and the underlying mechanisms of clothianidin resistant population of Frankliniella occidentalis to insecticides]. / 西花蓟马抗噻虫胺种群对杀虫剂的交互抗性及机制.

Clothianidin, belonging to neonicotinoid insecticide with systemic and contact mechanisms, is used to control the invasive pest Frankliniella occidentalis. To identify the resistance risk, we examined the cross-resistance to multiple insecticides and mechanisms of clothianidin resistant population of F. occidentalis. The results showed that F. occidentalis developed a high level of resis-tance to clothianidin (56.8-fold) after selecting for 45 generations. The resistant population of F. occidentalis had medium level of cross-resistance to thiamethoxam, imidacloprid, chlorpyrifos, cyhalothrin and emamectin benzoate (18.6>RR50>11.3), and the low level of cross-resistance to phoxim and methomyl, but no cross-resistance to chlorfenapyr and spinosad. The synergists piperonyl butoxide (PBO) and triphenyl phosphate (TPP) had significant synergistic effects on clothianidin in killing the resistant population (CL), Yunnan wild population (YN) and susceptible population (S). Compared with the sensitive population, the CL populations had significantly increased activities of mixed-functional oxidases P450(3.6-fold), b5(2.9-fold) and O-demethylase (4.9-fold), and carboxylesterase (2.5-fold), with no significant difference in the activities of glutathione S-transferases among CL and S populations. The results highlight the role of increasing mixed-functional oxidases and carboxylesterase in the resistance of F. occidentalis to clothianidin.

[Resistance risk and resistance stability of Frankliniella occidentalis to nitenpyram, clothianidin and thiamethoxam]. / 西花蓟马对烯啶虫胺、噻虫胺和噻虫嗪的抗性风险和抗性稳定性.

To effectively control the damage of Frankliniella occidentalis (Pergande), we evalutated the resistance risk and resistance stability of F. occidentalis to nitenpyram, clothianidin and thiamethoxam. With the method of dipping Phaseolus vuglaris, we selected the resistance populations from the susceptible population with nitenpyram, clothianidin and thiamethoxam, respectively. Both the resistance inheritance and resistance risk were analyzed with the resistance reality hereditary. After 30 generations' selections, the selected-populations showed high level of insecticide resistance to nitenpyram, clothianidin and thiamethoxam, with a resistance ratio of 44.7-fold, 45.5-fold, and 32.7-fold, respectively. The development rate of resistance to clothianidin, nitenpyram, and thiamethoxam reduced in turn, with a resistance reality heritability of 0.1503, 0.1336 and 0.1258, respectively. Stopping selection for 10 continuously generations, the resistance levels of selection resis-tance populations declined slowly, but could not regain the original susceptibility to nitenpyram, clothianidin and thiamethoxam. After resistance selection, the sensitivity of F. occidentalis nymphs to clothianidin, nitenpyram, and thiamethoxam was significantly higher than that of adults. F. occidentalis had the great potential to gain high level resistance to nitenpyram, clothianidin and thiamethoxam. Compared with other two insecticides, the resistance of F. occidentalis to thiamethoxam increased slower and decreased faster. Therefore, using thiamethoxam in nymph stage might be beneficial to effectively control F. occidentalis.

NlCYP4G76 and NlCYP4G115 Modulate Susceptibility to Desiccation and Insecticide Penetration Through Affecting Cuticular Hydrocarbon Biosynthesis in Nilaparvata lugens (Hemiptera: Delphacidae).

The functions of cuticular hydrocarbons (CHCs) are varied in insects, but one example is to reduce water loss. Previous work has suggested that biosynthesis of CHCs is strongly related to the CYP4G sub-family. Targeting these genes in the brown planthopper, Nilaparvata lugens Stål, might be a new application for integrated pest management. Therefore, we explored the functions of CYP4G76 (GenBank: KM217045.1) and CYP4G115 (GenBank: KM217046.1) genes in this study. The desiccation treatment (RH < 5%) for the duration of 1-3 days significantly increased the transcription level of CYP4G76 and CYP4G115. RNAi through the injection of CYP4G76 and CYP4G115 dsRNA could significantly decrease their expression, respectively, and further reduced the biosynthesis of CHCs, i.e., saturated and straight-chain alkanes. When CYP4G76 and CYP4G115 were suppressed, the susceptibility of N. lugens nymphs to desiccation increased, due to the deficiency of the CHCs in the insect's cuticle. When the expression of CYP4G76 and CYP4G115 was decreased, this resulted in an increased rate of penetration of the four insecticides: pymetrozine, imidacloprid, thiamethoxam and buprofezin. Therefore, CYP4G76 and CYP4G115 appear to regulate the biosynthesis of CHCs in N. lugens nymphs, which play a major role in protecting insects from water loss and the penetration of insecticides. CYP4G76 and CYP4G115 might be used as a novel target in integrated pest management to N. lugens.

Functional Characterization of Two Elongases of Very Long-Chain Fatty Acid from Tenebrio molitor L. (Coleoptera: Tenebrionidae).

The elongases of very long chain fatty acid (ELOVL or ELO) are essential in the biosynthesis of fatty acids longer than C14. Here, two ELO full-length cDNAs (TmELO1, TmELO2) from the yellow mealworm (Tenebrio molitor L.) were isolated and the functions were characterized. The open reading frame (ORF) lengths of TmELO1 and TmELO2 were 1005 bp and 972 bp, respectively and the corresponding peptide sequences each contained several conserved motifs including the histidine-box motif HXXHH. Phylogenetic analysis demonstrated high similarity with the ELO of Tribolium castaneum and Drosophila melanogaster. Both TmELO genes were expressed at various levels in eggs, 1st and 2nd instar larvae, mature larvae, pupae, male and female adults. Injection of dsTmELO1 but not dsTmELO2 RNA into mature larvae significantly increased mortality although RNAi did not produce any obvious changes in the fatty acid composition in the survivors. Heterologous expression of TmELO genes in yeast revealed that TmELO1 and TmELO2 function to synthesize long chain and very long chain fatty acids.

Aluminium-induced reduction of plant growth in alfalfa (Medicago sativa) is mediated by interrupting auxin transport and accumulation in roots.

The objective of this study was to investigate Al(3+)-induced IAA transport, distribution, and the relation of these two processes to Al(3+)-inhibition of root growth in alfalfa. Alfalfa seedlings with or without apical buds were exposed to 0 or 100 µM AlCl3 and were foliar sprayed with water or 6 mg L(-1) IAA. Aluminium stress resulted in disordered arrangement of cells, deformed cell shapes, altered cell structure, and a shorter length of the meristematic zone in root tips. Aluminium stress significantly decreased the IAA concentration in apical buds and root tips. The distribution of IAA fluorescence signals in root tips was disturbed, and the IAA transportation from shoot base to root tip was inhibited. The highest intensity of fluorescence signals was detected in the apical meristematic zone. Exogenous application of IAA markedly alleviated the Al(3+)-induced inhibition of root growth by increasing IAA accumulation and recovering the damaged cell structure in root tips. In addition, Al(3+) stress up-regulated expression of AUX1 and PIN2 genes. These results indicate that Al(3+)-induced reduction of root growth could be associated with the inhibitions of IAA synthesis in apical buds and IAA transportation in roots, as well as the imbalance of IAA distribution in root tips.

Gene Expression Analysis of Alfalfa Seedlings Response to Acid-Aluminum.

Acid-Aluminum (Al) is toxic to plants and greatly affects crop production worldwide. To understand the responses of plants to acid soils and Aluminum toxicity, we examined global gene expression using microarray data in alfalfa seedlings with the treatment of acid-Aluminum. 3,926 genes that were identified significantly up- or downregulated in response to Al3+ ions with pH 4.5 treatment, 66.33% of which were found in roots. Their functional categories were mainly involved with phytohormone regulation, reactive oxygen species, and transporters. Both gene ontology (GO) enrichment and KEGG analysis indicated that phenylpropanoid biosynthesis, phenylalanine metabolism, and flavonoid biosynthesis played a critical role on defense to Aluminum stress in alfalfa. In addition, we found that transcription factors such as the MYB and WRKY family proteins may be also involved in the regulation of reactive oxygen species reactions and flavonoid biosynthesis. Thus, the finding of global gene expression profile provided insights into the mechanisms of plant defense to acid-Al stress in alfalfa. Understanding the key regulatory genes and pathways would be advantageous for improving crop production not only in alfalfa but also in other crops under acid-Aluminum stress.

Brief Heat Stress Negatively Affects the Population Fitness and Host Feeding of Aphelinus asychis (Hymenoptera: Aphelinidae) Parasitizing Myzus persicae (Hemiptera: Aphididae).

Aphelinus asychis Walker (Hymenoptera: Aphelinidae), a polyphagous parasitoid, has been widely used as a biological control agent against Myzus persicae (Sulzer) (Hemiptera: Aphididae) and other aphid species. In order to ensure the successful biological control of M. persicae, we evaluated the influence of brief heat stresses (32.5, 35.0, 37.5, 40.0, and 41.5°C for 1 h), which occur frequently during the summer or under greenhouse conditions, on survival, longevity, host feeding, and parasitism of A. asychis that were reared on chili pepper at 25°C in the laboratory. Our results showed several major consequences on the parasitoid adults after brief heat stress. First, the survival of briefly heat stress-treated A. asychis female and male adults decreased significantly at ≥37.5°C and their longevity decreased as well. Second, the number of M. persicae nymphs infesting chili pepper killed through host feeding by the treated A. asychis females decreased significantly. Third, the cumulative number of mummified aphids and female progeny produced by the treated A. asychis also decreased significantly at ≥37.5°C. Our results indicated that a brief heat stress on A. asychis adults caused severe effects on major life history traits and total numbers of hosts killed by host feeding and parasitism, suggesting that high temperatures should be avoided to ensure the success of biological control of M. persicae and other aphid species using this parasitoid species.

Increase phosphorus availability from the use of alfalfa (Medicago sativa L) green manure in rice (Oryza sativa L.) agroecosystem.

Alfalfa is a good green manure source, but its effect on rice growth has not been fully elucidated. Two green manure species, alfalfa and broad bean (Vicia faba L.), and two N fertilizer levels, alone or combination, were applied to a rice field. The results indicated that alfalfa had more pronounced effects on increasing soil labile phosphorus (P) fractions (including NaHCO3-Pi, NaOH-Pi), P uptake and soil enzyme activities (dehydrogenase, urease, acid phosphatase and ß-glucosidase) than broad bean and N fertilizer. The transformation of NaHCO3-Po to labile P regulated by alfalfa played a significant direct and indirect effect on grain yield. Although a much lower N input from alfalfa addition, a similar grain yield with N fertilizer treatment was achieved, and the integration of alfalfa with N fertilizer produced the highest grain yield and P availability, which was associated with the highest urease, acid phosphatase and ß-glucosidase activity in soil. These results indicate that alfalfa green manure had a great ability of increasing grain yield through enhancing P availability in rice paddy, which could give us a way to reduce N fertilizer application by enhancing P availability.

[Resistance risk and resistance stability of Frankliniella occidentalis to imidacloprid, emamectin benzoate, and phoxim].

In order to effectively control the damage of Frankliniella occidentalis (Pergande), Phaseolus vuglaris was dipped with imidacloprid, phoxim, and emamectin benzoate, respectively to select the resistance populations of F. occidentalis from its susceptible population, and the resistance inheritance and resistance risk were analyzed with the resistance reality heredity. After 32, 32, and 24 generations' selection, the F. occidentalis populations obtained 13.8-fold, 29.4-fold and 39.0-fold resistance to imidacloprid, phoxim, and emamectin benzoate, respectively. The resistance reality heritability to imidacloprid, phoxim, and emamectin benzoate was 0.112, 0.166, and 0.259, respectively. The resistance development rate to emamectin benzoate was the fastest, followed by to phoxim, and to imidacloprid. The higher the resistance levels of the selected populations, the lower the differences between the larva and adult susceptibility to imidacloprid, phoxim, and emamectin benzoate. Stopping selection for 12 continuous generations, the resistance level of the selected resistance populations to imidacloprid, phoxim, and emamectin benzoate had definite decline, but it was difficult to regain the original susceptibility. F. occidentalis had a greater potential to gain high level resistance to imidacloprid, phoxim, and emamectin benzoate. Compared with the resistance of F. occidentalis to phoxim and emamectin benzoate, the resistance to imidacloprid increased slower and decreased faster, and thus, imidacloprid was more appropriate to control F. occidentalis in practice.

[Resistance mechanisms and cross-resistance of phoxim-resistant Frankliniella occidentalis Pergande population].

To understand the resistance risks of Frankliniella occidentalis Pergande against phoxim, this paper studied the resistance mechanisms of phoxim-resistant F. occidentalis population against phoxim and the cross-resistance of the population against other insecticides. The phoxim-resistant population had medium level cross-resistance to chlorpyrifos, lambda-cyhalothrin, and methomyl, low level cross-resistance to chlorfenapyr, imidacloprid, emamectin-benzoate, and spinosad, but no cross-resistance to acetamiprid and abamectin. The synergists piperonyl butoxide (PBO), s, s, s-tributyl phosphorotrithioate (DEF), and triphenyl phosphate (TPP) had significant synergism (P < 0.05) on the toxicity of phoxim to the resistant (XK), field (BJ), and susceptible (S) populations, while diethyl maleate (DEM) had no significant synergism to XK and S populations but had significant synergism to BJ population. As compared with S population, the XK and BJ populations had significantly increased activities of mixed-functional oxidases P450 (2.79-fold and 1.48-fold), b, (2.88-fold and 1.88-fold), O-demethylase (2.60-fold and 1.68-fold), and carboxylesterase (2.02-fold and 1.61-fold, respectively), and XK population had a significantly increased acetylcholine esterase activity (3.10-fold). Both XK and BJ population had an increased activity of glutathione S-transferases (1.11-fold and 1.20-fold, respectively), but the increment was not significant. The increased detoxification enzymes activities in F. occidentalis could play an important role in the resistance of the plant against phoxim.