Vacuolar ATPase subunit F is critical for larval survival in Henosepilachna vigintioctopunctata.

Vacuolar ATPase (vATPase) is an important proton pump in insect tissues including gut and Malpighian tubule. Subunit F, one of the 16 subunits of the vATPase holoenzyme, is not well characterized. Here, we found that two HvvATPaseF isoforms were highly expressed in the hindgut and Malpighian tubules (MT) in the 28-spotted lady-beetle Henosepilachna vigintioctopunctata, an agricultural pest that feeds on Solanaceae and Cucurbitaceae. Knockdown of both HvvATPaseF variants by RNA interference (RNAi) delayed larval growth and negatively affected ecdysis and adult emergence. In the midgut, RNAi treatment resulted in the disappearance of peritrophic membrane, the reduction in the size and the impaired integrity of the gut, which was associated with sparse principle cells and an increase in TUNEL- and EdU-positive cells. Whereas the MT were opaque and the tubule lumens were full of urine in dsegfp-fed larvae, the tubules were clear and the tubule lumens were empty in the dsvATPaseF-fed larvae. HvvATPaseF knockdown was also associated with a decrease in the abundance of the fat body and the levels of glucose, trehalose, triglyceride, total soluble amino acids and proteins, and an increase in glycogen. Consistent with the known effects of sugars on chitin formation, both the expression level of a chitin biosynthesis gene and the thickness of the head capsule cuticle were reduced in the HvvATPaseF-depleted beetles. Our results demonstrated that subunit F plays an essential role in H. vigintioctopunctata development.

Knockdown of Vacuolar ATPase Subunit G Gene Affects Larval Survival and Impaired Pupation and Adult Emergence in Henosepilachna vigintioctopunctata.

The vATPase holoenzyme consists of two functional subcomplexes, the cytoplasmic (peripheral) V1 and the membrane-embedded V0. Both V1 and V0 sectors contain eight subunits, with stoichiometry of A3B3CDE3FG3H in V1 and ac8c'c"def(Voa1p) in V0 respectively. However, the function of G subunit has not been characterized in any non-Drosophilid insect species. In the present paper, we uncovered that HvvATPaseG was actively transcribed from embryo to adult in a Coleopteran pest Henosepilachna vigintioctopunctata. Its mRNA levels peaked in larval hindgut and Malpighian tubules. RNA interference (RNAi)-mediated knockdown of HvvATPaseG significantly reduced larval feeding, affected chitin biosynthesis, destroyed midgut integrity, damaged midgut peritrophic membrane, and retarded larval growth. The function of Malpighian tubules was damaged, the contents of glucose, trehalose, lipid, total soluble amino acids and protein were lowered and the fat bodies were lessened in the HvvATPaseG RNAi larvae, compared with those in the PBS- and dsegfp-fed beetles. In contrast, the amount of glycogen was dramatically increased in the HvvATPaseG depletion ladybirds. As a result, the development was arrested, pupation was inhibited and adult emergence was impaired in the HvvATPaseG hypomorphs. Our results demonstrated that G subunit plays a critical role during larval development in H. vigintioctopunctata.

Integrated Microbiome-Metabolome Analysis Reveals Stage-Dependent Alterations in Bacterial Degradation of Aromatics in Leptinotarsa decemlineata.

To avoid potential harm during pupation, the Colorado potato beetle Leptinotarsa decemlineata lives in two different habitats throughout its developmental excursion, with the larva and adult settling on potato plants and the pupa in soil. Potato plants and agricultural soil contain a specific subset of aromatics. In the present study, we intended to determine whether the stage-specific bacterial flora plays a role in the catabolism of aromatics in L. decemlineata. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the operational taxonomic units (OTUs) obtained by sequencing of culture-independent 16S rRNA region enriched a group of bacterial genes involved in the elimination of mono- and polycyclic aromatics at the pupal stage compared with those at the larval and adult periods. Consistently, metabolome analysis revealed that dozens of monoaromatics such as styrene, benzoates, and phenols, polycyclic aromatics, for instance, naphthalene and steroids, were more abundant in the pupal sample. Moreover, a total of seven active pathways were uncovered in the pupal specimen. These ways were associated with the biodegradation of benzoate, 4-methoxybenzoate, fluorobenzoates, styrene, vanillin, benzamide, and naphthalene. In addition, the metabolomic profiles and the catabolism abilities were significantly different in the pupae where their bacteria were removed by a mixture of three antibiotics. Therefore, our data suggested the stage-dependent alterations in bacterial breakdown of aromatics in L. decemlineata.

The Leptinotarsa forkhead transcription factor O exerts a key function during larval-pupal-adult transition.

Forkhead box O (FoxO) protein, a major downstream transcription factor of insulin/insulin-like growth factor signaling/target of rapamycin pathway (IIS/TOR), is involved in the regulation of larval growth and the determination of organ size. FoxO also interacts with 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal transduction pathways, and hence is critical for larval development in holometabolans. However, whether FoxO plays a critical role during larval metamorphosis needs to be further determined in Leptinotarsa decemlineata. We found that 20E stimulated the expression of LdFoxO. RNA interference (RNAi)-aided knockdown of LdFoxO at the third-instar stage repressed 20E signaling and reduced larval weight. Although the resultant larvae survived through the third-fourth instar ecdysis, around 70% of the LdFoxO depleted moribund beetles developmentally arrested at prepupae stage. These LdFoxO depleted beetles were completely wrapped in the larval exuviae, gradually darkened and finally died. Moreover, approximately 12% of the LdFoxO RNAi beetles died as pharate adults. Ingestion of either 20E or JH by the LdFoxO depletion beetles excessively rescued the corresponding hormonal signals, but could not alleviate larval performance and restore defective phenotypes. Therefore, FoxO plays an important role in regulation of larval-pupal-adult transformation in L. decemlineata, in addition to mediation of IIS/TOR pathway and stimulation of ecdysteroidogenesis.

Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata.

Kynurenine pathway is critically important to catabolize tryptophan, to produce eye chromes, and to protect nervous system in insects. However, several issues related to tryptophan degradation remain to be clarified. In the present paper, we identified three genes (karmoisin, vermilion and cardinal) involved in kynurenine pathway in Henosepilachna vigintioctopunctata. The karmoisin and cardinal were highly expressed in the pupae and adults having compound eyes. Consistently, high-performance liquid chromatography result showed that three ommochrome peaks were present in adult heads rather than bodies (thoraces, legs, wings and abdomens). RNA interference (RNAi)-aided knockdown of vermilion caused accumulation of tryptophan in both adult heads and bodies, disappearance of ommochromes in the heads and a complete loss of eye color in both pupae and adults. Depletion of cardinal brought about excess of 3-hydroxykynurenine and insufficient ommochromes in the heads and decolored eyes. RNAi of karmoisin resulted in a decrease in ommochromes in the heads, and a partial loss of eye color. Moreover, a portion of karmoisin-, vermilion- or cardinal-silenced adults exhibited negative phototaxis, whereas control beetles showed positive phototaxis. Furthermore, dysfunctions of tryptophan catabolism impaired climbing ability. Our findings clearly illustrated several issues related to kynurenine pathway and provided a new insight into the physiological importance of tryptophan catabolism in H. vigintioctopunctata.

A switch of microbial flora coupled with ontogenetic niche shift in Leptinotarsa decemlineata.

In Leptinotarsa decemlineata, a final-instar wandering larva typically undergoes an ontogenetic niche shift (ONS), from potato plant during the foraging stage to its pupation site below ground. Using high-throughput sequencing of the bacterial 16S ribosomal RNA gene, we determined the hypothesis that the L. decemlineata pupae harbor stage-specific bacteria to meet the physiological requirements for underground habitat. We identified 34 bacterial phyla, comprising 73 classes, 208 orders, 375 families, and 766 genera in the collected specimens. Microbes across phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were enriched in the pupae, while those in the phylum Proteobacteria, Tenericutes, Firmicutes, and Bacteroidetes dominated in the larvae and adults. A total of 18 genera, including Blastococcus, Corynebacterium_1, Gordonia, Microbacterium, Nocardia, Nocardioides, Rhodococcus, Solirubrobacter, Tsukamurella, Enterococcus, Acinetobacter, Escherichia_Shigella, Lysobacter, Pseudomonas, and Stenotrophomonas, were specifically distributed in pupae. Moreover, soil sterilizing removed a major portion of bacteria in pupae. Specifically, both Enterococcus and Pseudomonas were eliminated in the soil sterilizing and antibiotic-fed beetle groups. Furthermore, the pupation rate and fresh pupal weight were similar, whereas the emergence rate and adult weight were decreased in the antibiotic-fed beetles, compared with controls. The results demonstrate that a switch of bacterial communities occurs in the pupae; the pupal-specific bacteria genera are mainly originated from soil; this bacterial biodiversity improves pupa performance in soil. Our results provide new insight into the evolutionary fitness of L. decemlineata to different environmental niches.

RNA interference targeting ecdysone receptor blocks the larval-pupal transition in Henosepilachna vigintioctopunctata.

Henosepilachna vigintioctopunctata is a serious insect pest which attacks a large number of nightshades and cucurbits in Asian countries, Brazil and Australia. Prolonged application of traditional pesticides has caused environmental pollution and exerted deleterious effects on human health. Finding new approaches with high target specificity and low environmental contamination has become an urgent task. RNA interference (RNAi) induced by double-stranded RNA (dsRNA) is expected to be applicable to managing this pest. Here we evaluated the effects of Escherichia coli-expressed dsRNAs targeting ecdysone receptor (EcR) gene via dietary delivery in laboratory and foliar spraying in a greenhouse. The target transcript was successfully knocked down when the 4th-instar larvae had fed on potato foliage dipped with dsEcR in a laboratory bioassay. Around 85% of the HvEcR RNAi larvae remained as prepupae or became abnormal pupae, and failed to emerge into adults. Ingestion of dsEcR-immersed foliage by the 3rd-instar larvae effectuated a comparable RNAi response and brought about more severe defects: all the resultant larvae arrested development, remained as prepupae and finally died. For assay in the greenhouse, a dsEcR-contained E. coli suspension was directly sprayed to the foliage of greenhouse-growing potato plants and the 3rd- and 4th-instar larvae were transferred to the leaves. High RNAi efficacy was obtained and identical RNAi phenotypes were observed in treated larvae. In addition, spraying dsEcR reduced leaf damage. Our results indicate a possibility of practical application of dsEcR as an environmentally friendly RNA pesticide to control H. vigintioctopunctata larvae.

Complete Genome Sequence of Stenotrophomonas maltophilia Strain CPBW01, Isolated from the Wings of the Colorado Potato Beetle in Xinjiang, China.

Bacteria of the genus Stenotrophomonas are opportunistic and have been documented in the guts of several insect species. Here, we present the complete genome sequence of S. maltophilia strain CPBW01, isolated from the wings of the Colorado potato beetle, Leptinotarsa decemlineata, collected from potato fields in Urumqi (43.71N, 87.39E), Xinjiang, China.