In silico promoter analysis and functional validation identify CmZFH, the co-regulator of hypoxia-responsive genes CmScylla and CmLPCAT.

Oxygen (O2) plays an essential role in aerobic organisms including terrestrial insects. Under hypoxic stress, the cowpea bruchid (Callosobruchus maculatus) ceases feeding and growth. However, larvae, particularly 4th instar larvae exhibit very high tolerance to hypoxia and can recover normal growth once brought to normoxia. To better understand the molecular mechanism that enables insects to cope with low O2 stress, we performed RNA-seq to distinguish hypoxia-responsive genes in midguts and subsequently identified potential common cis-elements in promoters of hypoxia-induced and -repressed genes, respectively. Selected elements were subjected to gel-shift and transient transfection assays to confirm their cis-regulatory function. Of these putative common cis-elements, AREB6 appeared to regulate the expression of CmLPCAT and CmScylla, two hypoxia-induced genes. CmZFH, the putative AREB6-binding protein, was hypoxia-inducible. Transient expression of CmZFH in Drosophila S2 cells activated CmLPCAT and CmScylla, and their induction was likely through interaction of CmZFH with AREB6. Binding to AREB6 was further confirmed by bacterially expressed CmZFH recombinant protein. Deletion analyses indicated that the N-terminal zinc-finger cluster of CmZFH was the key AREB6-binding domain. Through in silico and experimental exploration, we discovered novel transcriptional regulatory components associated with gene expression dynamics under hypoxia that facilitated insect survival.

Cytochrome P450s CYP380C6 and CYP380C9 in green peach aphid facilitate its adaptation to indole glucosinolate-mediated plant defense.

BACKGROUND: Overexpressing CIRCADIAN CLOCK ASSOCIATED1 in Arabidopsis thaliana (CCA1-ox) increases indole glucosinolate production and resistance to green peach aphid (Myzus persicae). Little is known of how aphids respond to this group of plant defense compounds or of the underlying molecular mechanism. RESULTS: Aphids reared on CCA1-ox for over 40 generations (namely the CCA population) became less susceptible to CCA1-ox than aphids maintained on the wild-type Col-0 (namely the COL population). This elevated tolerance was transgenerational as it remained for at least eight generations after the CCA population was transferred to Col-0. Intriguingly, transcriptome analysis indicated that all differential cytochrome P450 monooxygenase genes (MpCYPs), primarily MpCYP4s, MpCYP380s and MpCYP6s, were more highly expressed in the CCA population. Application of a P450 inhibitor to the CCA population resulted in decreased aphid reproduction on CCA1-ox, which was not observed if aphids were reared on Col-0. When indole glucosinolate biosynthesis in CCA1-ox was blocked using virus-induced gene silencing, the effect of the P450 inhibitor on the CCA population was attenuated, affirming the essential role played by MpCYPs in counteracting the defense mechanism in CCA1-ox that is low or absent in Col-0. Furthermore, we used host-induced gene silencing to identify MpCYP380C6 and MpCYP380C9 that specifically facilitated the CCA population to cope with CCA1-mediated plant defense. Expression profiles revealed their possible contribution to the transgenerational tolerance observed in aphids. CONCLUSION: MpCYP380C6 and MpCYP380C9 in aphids play a crucial role in mitigating indole glucosinolate-mediated plant defense, and this effect is transgenerational.

Aphid growth and reproduction on plants with altered sterol profiles: Novel insights using Arabidopsis mutant and overexpression lines.

Sterols are essential membrane components and are critical for many physiological processes in all eukaryotes. Insects and other arthropods are sterol auxotrophs that typically rely on a dietary source of sterols. Herbivorous insects generally obtain sterols from plants and then metabolize them into cholesterol, the dominant sterol in most insects. However, there is significant variation in phytosterol structure, and not all phytosterols are equally suitable for insects. In the current study, we used seven Arabidopsis thaliana lines that display altered sterol profiles due to mutations in the sterol biosynthetic pathway or to overexpression of key enzymes of the pathway, and investigated how plant sterol profiles affected green peach aphid (Myzus persicae) growth and reproduction. We also characterized the sterol profile of aphids reared on these Arabidopsis genotypes. Aphids on two mutant lines (14R/fk and ste1-1) that accumulated biosynthetic sterol intermediates (Δ8,14-sterols, and Δ7-sterols, respectively) all showed significantly reduced growth and reproduction. Aphids on SMT2COSUP plants (which have decreased ß-sitosterol but increased campesterol) also displayed significantly reduced growth and reproduction. However, aphids on SMT2OE plants (which have increased ß-sitosterol but decreased campesterol) performed similarly to aphids on wild-type plants. Finally, Arabidopsis plants that had an overproduction of sterols (CD-HMGROE) or decreased sterol esters (psat1-2) had no impact on aphid performance. Two noteworthy results come from the aphid sterol profile study. First, ß-sitosterol, cholesterol and stigmasterol were recovered in all aphids. Second, we did not detect Δ8,14-sterols in aphids reared on 14R/fk plants. We discuss the implications of our findings, including how aphid sterol content does not appear to reflect plant leaf sterol profiles. We also discuss the potential of modifying plant sterol profiles to control insect herbivore pests, including aphids.

Deleterious effects of electron beam irradiation on development and reproduction of tomato/potato psyllids, Bactericera cockerelli.

The potato/tomato psyllid Bactericera cockerelli causes serious damage to several solanaceous crops by direct feeding and vectoring Candidatus Liberibacter solanacearum, a bacterial pathogen. Electron beam (eBeam) irradiation is an environmentally friendly, chemical-free alternative method that is increasing in use for disinfestation of insect pests. We hypothesize that this irradiation technology will have detrimental effects on potato psyllid and thus impede its disease vectoring. To this end, we explored the effects of eBeam treatment ranging from 50 to 500 Gy on survival, development and reproduction of this pest. Impact on psyllids was apparently dose-dependent. When irradiated at 350 Gy, eggs could not hatch, 1st instar nymphs failed to emerge, and although a small portion of irradiated 5th instar nymphs survived, the emerged adults were mostly deformed. Abnormality in eclosed adults suggests harmful effects of eBeam on metamorphosis. Reproduction was seriously impaired when female psyllids were exposed to eBeam at the 5th instar nymphal or young adult stage, presumably due to inability to form oocytes. In addition, reciprocal crosses between irradiated and untreated psyllids indicated that female psyllids were more radiosensitive than males to eBeam. Taken together, these findings indicate that eBeam negatively impacted potato psyllid development and reproduction, which would inevitably compromise its disease transmission capacity. A dose of 350 Gy can be considered as a reference dose for effective control of potato psyllids.