Effects of periodic heat events on the reproduction and longevity of female and male Agasicles hygrophila (Coleoptera: Chrysomelidae).

Alternanthera philoxeroides (Amaranthaceae), commonly known as alligator weed, is a globally invasive and detrimental perennial weed. Agasicles hygrophila serves as an important biocontrol agent for alligator weeds. However, during mid-summer, when temperatures increase, A. hygrophila populations experience a significant decline, leading to ineffective weed control. This study has examined the impact of periodic heat events on the reproduction and survival of A. hygrophila females and males using various mating combinations and durations of temperature treatments. The results demonstrated significant effects on all of the studied parameters across mating combinations when compared with the control. Under the same temperature combination, the fecundity and survival rates of females, as well as the egg-hatching rate, decreased significantly with increasing repeated heat exposure. Furthermore, the egg-hatching rate varied significantly among different temperatures and time-interval combinations. In addition, the females displayed greater sensitivity to heat stress than males in terms of fecundity. These findings enhance our understanding of A. hygrophila population dynamics during summer and provide insights into the release of biocontrol agents in diverse regions with varying climates.

Host-Plant Selection Behavior of Ophraella communa, a Biocontrol Agent of the Invasive Common Ragweed Ambrosia artemisiifolia.

Understanding the host-selection behavior of herbivorous insects is important to clarify their efficacy and safety as biocontrol agents. To explore the host-plant selection of the beetle Ophraella communa, a natural enemy of the alien invasive common ragweed (Ambrosia artemisiifolia), we conducted a series of outdoor choice experiments in cages in 2010 and in open fields in 2010 and 2011 to determine the preference of O. communa for A. artemisiifolia and three non-target plant species: sunflower (Helianthus annuus), cocklebur (Xanthium sibiricum), and giant ragweed (Ambrosia trifida). In the outdoor cage experiment, no eggs were found on sunflowers, and O. communa adults rapidly moved from sunflowers to the other three plant species. Instead, adults preferred to lay eggs on A. artemisiifolia, followed by X. sibiricum and A. trifida, although very few eggs were observed on A. trifida. Observing the host-plant selection of O. communa in an open sunflower field, we found that O. communa adults always chose A. artemisiifolia for feeding and egg laying. Although several adults (<0.02 adults/plant) stayed on H. annuus, no feeding or oviposition were observed, and adults quickly transferred to A. artemisiifolia. In 2010 and 2011, 3 egg masses (96 eggs) were observed on sunflowers, but they failed to hatch or develop into adults. In addition, some O. communa adults crossed the barrier formed by H. annuus to feed and oviposit on A. artemisiifolia planted in the periphery, and persisted in patches of different densities. Additionally, only 10% of O. communa adults chose to feed and oviposit on the X. sibiricum barrier. These findings suggest that O. communa poses no threat to the biosafety of H. anunuus and A. trifida and exhibits a robust dispersal capacity to find and feed on A. artemisiifolia. However, X. sibiricum has the potential to be an alternative host plant for O. communa.

Cold Acclimation and Supercooling Capacity of Agasicles hygrophila Adults.

Agasicles hygrophila Selman and Vogt is used in the biological control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, with the northward establishment of A. philoxeroides in China, the weak adaptivity of A. hygrophila to cold weather has resulted in the ineffective control of A. philoxeroides in northern China. Cold acclimation can significantly enhance insect cold tolerance, enabling them to cope with more frequent climate fluctuations. To improve the biological control efficacy of A. hygrophila in cold climates, we compared the effects of rapid cold hardening and acclimation on A. hygrophila under laboratory conditions. On initially transferring adults from 26 to -10 °C for 2 h, mortality reached 80%. However, when pre-exposed to 0 °C for 2 h and then transferred to -10 °C for 2 h, adult mortality was reduced to 36.67%. These findings indicate that cold acclimation can enhance the cold tolerance of A. hygrophila under laboratory conditions. However, the beneficial cold acclimation effects waned after more than 15 min of recovery at 26 °C. Exposure to 15 °C for 24 h or gradual cooling from 0 to -10 °C at 1 °C·min-1 also induced cold acclimation, indicating that long-term cold and fluctuating cold acclimation are also potentially effective strategies for enhancing low-temperature tolerance.

Regulatory Mechanism of Transcription Factor AhHsf Modulates AhHsp70 Transcriptional Expression Enhancing Heat Tolerance in Agasicles hygrophila (Coleoptera: Chrysomelidae).

Agasicles hygrophila is a classical biological agent used to control alligator weed (Alternanthera philoxeroides). Previous research has indicated that the heat shock factor (HSF) is involved in regulating the transcriptional expression of Hsp70 in response to heat resistance in A. hygrophila. However, the regulatory mechanism by which AhHsf regulates the expression of AhHsp70 remains largely unknown. Here, we identified and cloned a 944 bp AhHsp70 promoter (AhHsp70p) region from A. hygrophila. Subsequent bioinformatics analysis revealed that the AhHsp70p sequence contains multiple functional elements and has a common TATA box approximately 30 bp upstream of the transcription start site, with transcription commencing at a purine base approximately 137 bp upstream of ATG. Promoter deletion analyses revealed that the sequence from -944 to -744 bp was the core regulatory region. A dual-luciferase reporter assay indicated that overexpressed AhHsf significantly enhanced the activity of AhHsp70p. Furthermore, qPCR showed that AhHsp70 expression increased with time in Spodoptera frugiperda (Sf9) cells, and AhHsf overexpression significantly upregulated AhHsp70 expression in vitro. Characterization of the upstream regulatory mechanisms demonstrated that AhHsf binds to upstream cis-acting elements in the promoter region of AhHsp70 from -944 to -744 bp to activate the AhHSF-AhHSP pathway at the transcriptional level to protect A. hygrophila from high temperature damage. Furthermore, we proposed a molecular model of AhHsf modulation of AhHsp70 transcription following heat shock in A. hygrophila. The findings of this study suggest that enhancing the heat tolerance of A. hygrophila by modulating the upstream pathways of the Hsp family can improve the biocontrol of A. philoxeroides.

Heat Shock Factor Is Involved in Regulating the Transcriptional Expression of Two Potential Hsps (AhHsp70 and AhsHsp21) and Its Role in Heat Shock Response of Agasicles hygrophila.

Heat shock proteins are molecular chaperones that are involved in numerous normal cellular processes and stress responses, and heat shock factors are transcriptional activators of heat shock proteins. Heat shock factors and heat shock proteins are coordinated in various biological processes. The regulatory function of heat shock factors in the expression of genes encoding heat shock proteins (Hsps) has been documented in some model insects, however, the role of transcription factors in modulating Hsps in other insects is still limited. In this study, one heat shock factor gene (AhHsf) was isolated and its two potential target genes (AhHsp70 and AhsHsp21) were confirmed from Agasicles hygrophila. AhHsf sequence analysis indicated that it belongs to the Hsfs gene family. RT-qPCR showed that expression levels of heat shock factors and of two heat shock proteins significantly increased under heat stress. Injection with double-stranded Hsf RNA in freshly emerged adult beetles significantly inhibited expression of AhHsp70 and AhsHsp21, shortened the adult survival, drastically reduced egg production, and ultimately led to a decrease in fecundity. RNA interference (RNAi)-mediated suppression of AhHsp70 or AhsHsp21 expression also significantly affected expression of AhHsf. Our findings revealed a potential transcriptional function of AhHsf to regulate expression of AhHsp70 and AhsHsp21, which may play a key role in A. hygrophila thermotolerance. Our results improve our understanding of the molecular mechanisms of the AhHsf - AhHsps signaling pathway in A. hygrophila.

Identification and Characterization of the Vitellogenin Receptor Gene and Its Role in Reproduction in the Alligatorweed Flea Beetle, Agasicles hygrophila.

Agasicles hygrophila is an effective biological control agent for the invasive weed Alternanthera philoxeroides, and because of this it has been introduced to many parts of the world where A. philoxeroides is a problem. Despite this, there are no reports at present about the reproduction of this important insect. Vitellogenin receptors (VgRs) belong to the superfamily of low-density lipoprotein receptors (LDLRs). One of the roles of VgRs is to regulate the absorption of yolk protein in insects. In this study, the full length vitellogenin receptor gene (AhVgR) from A. hygrophila was sequenced and found to encode a predicted protein of 1,642 amino acids. Sequence analysis of AhVgR revealed that it contains conserved structural motifs common to LDLR family members, and a phylogenetic analysis placed AhVgR as a separate group among the order Coleoptera. AhVgR was found to be specifically expressed in ovarian tissues, and it is first transcribed in the newly-emerged females. The expression patterns are consistent with VgR genes in other insects. RNA interference (RNAi)-mediated suppression of AhVgR gene expression in adult A. hygrophila females inhibited yolk protein deposition in the ovaries, shortened the ovariole, drastically reduced egg production, and ultimately led to a decrease in fecundity. In summary, our work shows that AhVgR is critical for transporting Vg into the oocytes and plays an important role in A. hygrophila reproduction.

Identification and Expression Patterns of Three Vitellogenin Genes and Their Roles in Reproduction of the Alligatorweed Flea Beetle Agasicles hygrophila (Coleoptera: Chrysomelidae).

The alligatorweed flea beetle Agasicles hygrophila is an insect used for biological control of the aquatic weed Alternanthera philoxeroides (alligatorweed). Because these insects are oviparous, synthesis, and transportation of yolk proteins is integral to reproduction. Vitellin, the chief protein constituent in egg yolk, is mainly synthesized in the fat body and its synthesis is regulated by the transcript levels of Vitellogenin (Vg). In our study, we first cloned and characterized three Vg genes from A. hygrophila and quantified the expression levels of these Vgs in different tissues and developmental stages by RT-qPCR. Analysis of the full-length cDNA sequences of the three A. hygrophila Vg genes revealed that the open reading frames of AhVg1, AhVg2, and AhVg3 were 5175, 5346, and 5385 bp, encoding 1724, 1781, and 1794 amino acids, respectively. RT-qPCR analysis revealed that these three AhVgs have similar expression patterns; expression in the fat body was significantly higher than that in other tissues, and the highest expression was observed in the adult developmental stage. RNA interference was used to explore the functions of the AhVgs. A. hygrophila female adults injected with dsRNA targeting the AhVg genes showed decreased AhVg gene expression. Down regulation of all three AhVgs significantly affected ovary development, reduced egg laying capacity, and reduced the egg hatch rate compared with the control groups. Our findings provide the basis for further study of the functions of Vg genes in other insect species.

Induced Thermotolerance and Expression of Three Key Hsp Genes (Hsp70, Hsp21, and sHsp21) and Their Roles in the High Temperature Tolerance of Agasicles hygrophila.

Thermal adaptation plays a fundamental role in the expansion and distribution of insects, and heat shock proteins (Hsps) play important roles in the temperature adaptation of various organisms. To determine the roles of Hsp genes (Hsp70, Hsp21, and sHsp21) on the high temperature tolerance of Agasicles hygrophila, we obtained complete cDNA (complementary DNA) sequences for Hsp70, Hsp21, and sHsp21 by rapid amplification of cDNA ends (RACE), analyzed their expression profiles under different high temperature treatments by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and performed functional verification by RNA interference (RNAi). The open reading frames of Hsp70, Hsp21, and sHsp21 were 1940, 543, and 567 bp, encoding 650, 180, and 188 amino acids, respectively. Their molecular weights (MWs) were 71.757, 20.879, and 21.510 kDa, and the isoelectric points were 5.63, 6.45, and 6.24, respectively. Phylogenetic tree analysis showed that the Hsp70, Hsp21, and sHsp21 genes of A. hygrophila were relatively conserved in evolution. The Hsp70 and Hsp21 genes in A. hygrophila were homologous to those in Leptinotarsa decemlineata (87 and 79% similarity, respectively), and the sHsp21 gene in A. hygrophila was homologous to that in Lissorhoptrus oryzophilus (74% similarity). The amino acid polypeptide chain had highly conserved sequences of DLGGGTFD, VLVGGSTR, and GPTIEEVD. The sequence of EEVD was the characteristic motif of cytoplasmic Hsp70, and the highly conserved sequences of MALFR and MSLLP were characteristic sequences of Hsp2 and sHsp21, respectively. Relative quantitative real time PCR showed that the three Hsps could be induced by 4-h treatment at high temperatures. Significant upregulation of these Hsps was observed when the temperature was further increased. The RNAi results showed that the injection of the three Hsps' dsRNA could suppress the expression at the gene level significantly. Compared with the control group, high temperature heat shock reduced the fecundity of A. hygrophila significantly, and the fecundity decreased with the increase in temperature. Our results suggest that Hsp70, Hsp21, and sHsp21 might play key roles in high temperature adaptation of A. hygrophila and help improve our understanding of their mechanism of thermotolerance.