Heat Shock Protein 70 Genes Are Involved in the Thermal Tolerance of Hippodamia variegata.

Previous studies have shown that the survival and reproduction of Hippodamia variegata are increasingly harmed by progressive increases in temperature (from 32 °C to 35 °C and 38 °C). In this study, transcriptome sequencing analysis was performed on H. variegata, after being exposed to different temperatures (from 32 to 38 °C) for 24 h, using high-throughput sequencing technology. We found the largest number of differentially expressed genes (DEGs) in the 35 °C vs. 32 °C group (1151) followed by the 38 °C vs. 32 °C group (1054) and then the 38 °C vs. 35 °C group (901), indicating that H. variegata expressed the largest number of newly mobilized genes under medium-high temperature (35 °C). Gene functional analysis showed that a large number of DEGs were involved in "Catalytic activity", "Oxidoreductase activity", "Metabolic pathways", and "Longevity regulating pathway-multiple species" gene groups. We randomly selected nine DEGs for validation using qRT-PCR. The results of qRT-PCR were consistent with the transcriptome data, confirming their reliability. Finally, the RNAi results showed that adult survival, longevity, and fecundity were lower in the group in which gene expression of the heat shock proteins (Hsp70-01 and Hsp68) was suppressed than in the control group (injection ds-GFP) at all the experimental temperatures (32, 35, and 38 °C). Our results indicate the important role of the heat shock proteins (Hsp70-01 and Hsp68) in resistance to high-temperature stress in H. variegata and provide a molecular basis for analyzing its thermotolerance mechanism.

Two Structural Analogs of Kairomones are Detected by an Odorant Receptor HvarOR28 in the Coccinellid Hippodamia variegata.

In natural environments, general plant volatiles and herbivore-induced plant volatiles (HIPVs) serve as critical clues for predatory natural enemies in the search for prey. The insect olfactory system plays a vital role in perceiving plant volatiles including HIPVs. In this study, we found that HIPV (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) and the plant volatile geranyl acetate (GA), two structurally similar chemicals, displayed electrophysiological activities on the antennae of the ladybird Hippodamia variegata, but were only attractive to adult females in behavior. Moreover, mated female ladybirds laid a significantly higher number of eggs on TMTT-treated and GA-treated cotton leaves compared to controls. Screening of female-biased odorant receptors (ORs) from the antennal transcriptomes, performing Xenopus oocytes expression coupled with two-electrode voltage clamp recordings, suggested that HvarOR28 specifically tuned to TMTT and GA. Molecular docking and site-directed mutagenesis revealed that the amino acid residues Tyr143 and Phe81 of HvarOR28 are the key site for binding with TMTT and GA. Furthermore, RNA interference (RNAi) assay demonstrated that HvarOR28-silenced individuals demonstrated a notable decrease in electrophysiological responses, even female adults almost lost behavioral preference for the two compounds. Thus, it could be concluded that HvarOR28 in H. variegata contributes to facilitating egg laying through the perception of TMTT and GA. These findings may help to develop new olfactory modulators based on the behaviorally active ligands of HvarOR28.

Impact of Aphis pomi-tending Formica rufa (Hymenoptera: Formicidae) on biological parameters of Hippodamia variegata (Coleoptera: Coccinellidae).

One of the key reasons for the poor performance of natural enemies of honeydew-producing insect pests is mutualism between ants and some aphid species. The findings demonstrated that red wood ant, Formica rufa Linnaeus (Hymenoptera: Formicidae) had a deleterious impact on different biological parameters of the lady beetle, Hippodamia variegata Goeze (Coleoptera: Coccinellidae). H. variegata laid far fewer eggs in ant-tended aphid colonies, laying nearly 2.5 times more eggs in ant absence. Ants antennated and bit the lady beetle eggs, resulting in significantly low egg hatching of 66 per cent over 85 per cent in ant absent treatments. The presence of ants significantly reduced the development of all larval instars. The highest reduction was found in the fourth larval instar (31.33% reduction), and the lowest in the first larval instar (20% reduction). Later larval instars were more aggressively attacked by ants than earlier instars. The first and second larval instars stopped their feeding and movement in response to ant aggression. The third and fourth larval instars modified their mobility, resulting in increased ant aggression towards them. Adult lady beetles were shown to be more vulnerable to ant attacks than larvae. However, H. variegata adults demonstrated counterattacks in the form of diverse defensive reaction behaviours in response to F. rufa aggression.

Impact of diapause on the cuticular hydrocarbons and physiological status of Hippodamia variegata.

The spotted amber ladybird, Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae), is known to be a potent predator of aphids, psyllids, whiteflies, mealybugs, and some butterfly species. This ladybeetle overwinters in the diapausing adult stage. The current study aimed to evaluate the impact of diapause on the energy resources and cuticular hydrocarbons (CHCs) of the female ladybeetle, specifically comparing the changes in glycogen, lipid, and protein contents, and CHCs profile of diapausing and non-diapausing adults. In this study, gas chromatography-mass was used to analyze whole-body extracts of the beetles. Results showed no significant differences between the amount of glycogen, lipid, and protein contents of diapausing and non-diapausing ladybeetle. The CHCs profile of H. variegata consisted of 24 hydrocarbons categorized into 2 groups: linear aliphatic hydrocarbons (n-alkanes) and methyl-branched hydrocarbons (17 molecules), as well as unsaturated cyclic compounds (7 molecules). The n-alkanes, with 14 compounds, were identified as the primary constituents of the CHCs of the ladybeetle. Six molecules were common to non-diapausing and diapausing beetles, 5 were exclusive to non-diapausing beetles, and 13 were exclusive to diapausing beetles. Moreover, we noted a significant difference in the quantity and quality of CHCs between diapausing and non-diapausing beetles, with diapausing beetles synthesizing more CHCs with longer chains. This disparity in CHC profiles was concluded to be an adaptation of H. variegata to survive harsh environmental conditions during diapause.

Recruitment of Hippodamia variegata by active volatiles from Glycyrrhiza uralensis and Alhagi sparsifolia plants infested with Aphis atrata.

BACKGROUND: Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae), a dominant predatory natural enemy species in cotton-planting, is a key biological control agent for aphids in China. Our previous study showed that herbivore-induced plant volatiles (HIPVs) from Glycyrrhiza uralensis (Fisch.) (Fabales: Fabaceae) and Alhagi sparsifolia (Desv.) (Fabales: Fabaceae) plants infested with Aphis atrata (Zhang) (Homoptera: Aphididae), were important semiochemicals for Hippodamia variegata to locate aphids. However, little was known about the varieties and function of active volatiles from HIPVs of the two plant species. RESULTS: In this study, results from gas chromatography-electroantennography detection (GC-EAD) demonstrated that seven HIPVs (butyl acrylate, α-pinene, butyl isobutyrate, ß-pinene, butyl butyrate, 1,3-diethylbenzene and 1,4-diethylbenzene) identified from the two damaged plant species elicited antennal responses from Hippodamia variegata. Also, results from gas chromatograph-mass spectrometry (GC-MS) analysis showed that the concentrations of the seven active volatiles were significantly higher than those from corresponding healthy plants. Hippodamia variegata exhibited varying degrees of response to each active volatile in electroantennography (EAG) trials, however, only α-pinene, butyl isobutyrate, ß-pinene and butyl butyrate significantly attracted Hippodamia variegata in behavioral trials conducted in the laboratory. They also had a better trapping effect on Hippodamia variegata in cotton fields. CONCLUSION: Four active compounds (α-pinene, butyl isobutyrate, ß-pinene and butyl butyrate) identified from two damaged plant species were considered the most effective HIPVs that attract Hippodamia variegata. These findings provide possibilities for the development of Hippodamia variegata attractants. They also provide a theoretical basis for the biological prevention and control of aphids using Hippodamia variegata. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Odorant-Binding Protein HvarOBP5 in Ladybird Hippodamia variegata Regulates the Perception of Semiochemicals from Preys and Habitat Plants.

A total of 12 OBPs were identified in the antennae of ladybird Hippodamia variegata. HvarOBP1/2/5/6/10/11 were highly expressed in adults, whereas HvarOBP3/4/7/8/9/12 had higher expression levels in larvae. In adults, HvarOBP1/2/4/5/6/7/8/11/12 were highly expressed in antennae. Among these, recombinant HvarOBP5 strong bound with (E)-ß-farnesene (EßF), (R)-(+)-limonene, (E,E)-4,8,12-trimethyltrideca-l,3,7,11-tetraene (TMTT), (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), hexyl hexanoate, and geranyl acetate. Molecular docking indicated that Leu42, Lys43, and His64 were the key binding sites of HvarOBP5. All six ligands evoked electroantennography (EAG) responses in ladybirds. Moreover, (R)-(+)-limonene and hexyl hexanoate were attractive to both sexes. After RNA interference for 72 h, the EAGs of dsRNA-injected ladybirds to DMNT and hexyl hexanoate were significantly decreased by 73.8 and 78.6%, respectively. Both dsRNA-injected males and females showed significantly lower behavioral preferences for DMNT and hexyl hexanoate. These findings suggest that HvarOBP5 in H. variegata plays an important role in the perception of semiochemical cues from preys and habitat plants.

The supercooling point depression is the leading cold tolerance strategy for the variegated ladybug, [Hippodamia variegata (Goezel)].

The variegated ladybug, Hippodamia variegata is one of the most effective predators of various pests that hibernate as adult beetles. During the overwintering period from April 2021 to March 2022, we examined the supercooling point (SCP), cold tolerance, and physiological adaptations of beetles in Kerman, Iran. The beetles exhibited their greatest cold tolerance (63.4% after 24 h at -5°C) when their SCP was lowest (-23.2°C). Conversely, from April to October 2021, the SCP reached its peak (approximately -13.0°C), while cold tolerance was at its lowest level (6.7% after 24 h at -5°C). Cryoprotectant content (trehalose, glycerol, and glucose) was at its highest level in September (11.15, 10.82, and 6.31 mg/g, respectively). The critical thermal minimum (CTmin) reached its lowest point of -2.2°C in January and February. The lowest point of the lower lethal temperature (LLT) coincided with the lowest level of the SCP and the highest level of cold tolerance (in February, LT50 = -5.3°C, SCP = -23.2°C, and survival = 77.78% at -4°C/24 h). Chill-coma recovery time (CCRT) was examined at five different temperatures and two different exposure durations. The CCRT increased with a decrease in exposure temperature and time (68.0 s at -2°C after 2 h and 102.0 s at -2°C after 4 h). As the majority of the overwintering beetle's mortality occurred at temperatures significantly higher than SCP, the adults of H. variegata are chill-susceptible insects that primarily rely on a depressed supercooling point to cope with unfavorable conditions during the overwintering period.

Antenna-Biased Odorant Receptor HvarOR25 in Hippodamia variegata Tuned to Allelochemicals from Hosts and Habitat Involved in Perceiving Preys.

Odorant receptors (ORs) of ladybird Hippodamia variegata play vital chemosensory roles in searching and locating preys. In the current study, 37 ORs were initially identified from the antennal transcriptome of H. variegata. The quantitative polymerase chain reaction demonstrated that several HvarORs including HvarOR25 were specific or enriched in ladybird antennae. In two-electrode voltage clamp recordings, recombinant HvarOR25 was narrowly tuned to six chemical ligands including aphid-induced, aphid-derived, and plant-derived volatiles. In electroantennogram assays, all six volatiles elicited electrophysiological responses. Among the six volatiles, cis-3-hexenyl acetate, hexyl butyrate, hexyl hexanoate, and 3-methyl-3-buten-1-ol were attractive for both sexes of H. variegata. Additionally, molecular docking indicated that HvarOR25 was bound to all ligands with high binding affinities. Taken together, HvarOR25 facilitates perception of preys by recognizing relevant allelochemicals from hosts and habitat. Our findings provide valuable insights into understanding biological functions of HvarORs and help to develop a novel biocontrol strategy based on olfactory-active compounds.

Species Complex and Temporal Associations between Coccinellids and Aphids in Alfalfa Stands in Spain.

Alfalfa is known to be an important reservoir harboring natural enemies. The reduction in insecticide sprayings in recent years has allowed us to study the coccinellid species complex in this crop and the relationship between these predators and aphids. Alfalfa was sampled by sweep-netting throughout its productive period in several commercial stands each year between 2010 and 2021. The numbers and species of aphids and coccinellids were recorded. Sixteen coccinellid species were found. Coccinella septempunctata and Hippodamia variegata were, by far, the most prevalent species, with the former dominating during the first and second intercuts, whereas the latter dominated from the third to the fifth intercut. Acyrthosiphon pisum and Therioaphis trifolii were the most abundant aphid species, peaking in the second and fourth intercuts, respectively. Positive correlations were found between the abundance of C. septempunctata and A. pisum at the second intercut, between H. variegata and T. trifolii at the fourth intercut, and between H. variegata and the total number of aphids in the fifth intercut. This study helps to increase the knowledge on the predator-prey relationships of this crop and allows for designing strategies of conservation biological control against aphids.

Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis. Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder), eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-ß, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.

Satellitome Analysis in the Ladybird Beetle Hippodamia variegata (Coleoptera, Coccinellidae).

Hippodamia variegata is one of the most commercialized ladybirds used for the biological control of aphid pest species in many economically important crops. This species is the first Coccinellidae whose satellitome has been studied by applying new sequencing technologies and bioinformatics tools. We found that 47% of the H. variegata genome is composed of repeated sequences. We identified 30 satellite DNA (satDNA) families with a median intragenomic divergence of 5.75% and A+T content between 45.6% and 74.7%. This species shows satDNA families with highly variable sizes although the most common size is 100-200 bp. However, we highlight the existence of a satDNA family with a repeat unit of 2 kb, the largest repeat unit described in Coleoptera. PCR amplifications for fluorescence in situ hybridization (FISH) probe generation were performed for the four most abundant satDNA families. FISH with the most abundant satDNA family as a probe shows its pericentromeric location on all chromosomes. This location is coincident with the heterochromatin revealed by C-banding and DAPI staining, also analyzed in this work. Hybridization signals for other satDNA families were located only on certain bivalents and the X chromosome. These satDNAs could be very useful as chromosomal markers due to their reduced location.

Effects of exposure to imidacloprid direct and poisoned cotton aphids Aphis gossypii on ladybird Hippodamia variegata feeding behavior.

Imidacloprid is a pesticide used to control aphid infestations of cotton plants. However, poisoned aphids also serve as food for the ladybird natural predator Hippodamia variegata. We investigated whether imidacloprid-treated eggs, pupae, and adults of H. variegata and poisoned aphids altered ladybird predatory behavior. Laboratory bioassay results demonstrated that 0.72 g/L imidacloprid was lethal to ladybirds. Imidacloprid significantly reduced the hatching and emergence rates of H. variegata, and these effects were time and dose dependent. Predation was most adversely affected when the ladybirds directly consumed poisoned aphids and less so when directly exposed to the insecticide at sublethal concentrations. Imidacloprid use in cotton fields should be restricted to the initial stages of aphid infestation to avoid the period when adult ladybirds are present.

Lethal and sub-lethal effects of imidacloprid on the aphidophagous coccinellid hippodamia variegata.

Hippodamia variegata is one of the most abundant ladybird species in Greece, preying on several aphid species and other arthropods, of which many are pests of cultivated plants. Imidacloprid, a neonicotinoid insecticide, is commonly used for controlling sucking insects; at the same time, however, it can cause various sub-lethal effects on non-target organisms. The development of IPM programs against pests requires an evaluation of the side effects of insecticides on natural enemies. We evaluated the sub-lethal effects of imidacloprid on H. variegata. Our results demonstrate that imidacloprid at a LC10 (3.92 mg (a.i.) L-1 and LC30 (8.69 mg (a.i.) L-1) decreased adult longevity and survival rate. In addition, demographic parameters, such as the intrinsic rate of increase (r), finite rate of increase (λ) and net reproductive rate (R0), were reduced when exposed to sub-lethal concentrations of imidacloprid. These results demonstrate a negative influence of imidacloprid at sub-lethal concentrations on H. variegata which could reduce biological control services provided by this predator.

Protective effects of the egg stalk of Paratrioza sinica (Hemiptera: Psyllidae) at various angles and spacings against three predaceous coccinellids, Harmonia axyridis, Coccinella septempunctata and Hippodamia variegata (Coleoptera: Coccinellidae).

BACKGROUND: Paratrioza sinica is a major pest of wolfberry (Lycium barbarum). Coccinellids can effectively control various developmental stages of P. sinica but not the stalked egg. To investigate the protective role of egg stalks against the predaceous coccinellids Harmonia axyridis, Coccinella septempunctata and Hippodamia variegata, we studied the functional responses to, and effects of, two factors: the angle between the egg stalk and the leaf plane, and the spacing between egg stalks. RESULTS: The searching rate, handling time and theoretical maximum egg consumption of H. variegata were optimal among the three ladybug species. Egg consumption by coccinellids was maximum and minimum at stalk angles of 0° and 90°, respectively. The average reduction in the rate of egg consumption when the stalk angle was 90° relative to 0° was significantly smaller for larvae of coccinellids compared with adults, and for H. variegata compared with the other two species. Optimal spacing of eggs for consumption by predators varied with the predator species and their developmental stage, and was close to the body length of the predators. CONCLUSION: The egg stalk serves as a physical protection against predators. The evolutionary selective advantage of the egg stalk in providing protection against predators requires more attention. Appropriate selection of predators and irrigation strategies may improve control of P. sinica. © 2017 Society of Chemical Industry.

Estimating the effect of plant-provided food supplements on pest consumption by omnivorous predators: lessons from two coccinellid beetles.

BACKGROUND: Plant-provided food supplements can influence biological pest control by omnivorous predators in two counteracting ways: they can (i) enhance predator populations, but (ii) reduce pest consumption by individual predators. Yet the majority of studies address only one of these aspects. Here, we first tested the influence of canola (Brassica napus L.) pollen supplements on the life history of two ladybeetle species: Hoppodamia variegata (Goeze) and Coccinella septempunctata (L.). We then developed a theoretical model to simulate total pest consumption in the presence and absence of pollen supplements. RESULTS: Supplementing a prey diet with canola pollen increased H. variegata larval survival from 50 to 82%, and C. septempunctata female oviposition by 1.6-fold. Model simulations revealed a greater benefit of pollen supplements when relying on C. septempunctata for pest suppression than on H. variegata. CONCLUSION: For these two predators, the tested pollen serves as an essential supplement to a diet of prey. However, the benefit of a mixed prey-pollen diet was not always sufficient to overcome individual decrease in pest consumption. Taken together, our study highlights the importance of addressing both positive and negative roles of plant-provided food supplements in considering the outcome for biological control efforts that rely on omnivorous predators. © 2016 Society of Chemical Industry.

Intraguild Predation Responses in Two Aphidophagous Coccinellids Identify Differences among Juvenile Stages and Aphid Densities.

(1) Intraguild predation (IGP) can occur among aphidophagous predators thus reducing their effectiveness in controlling crop pests. Among ladybirds, Coccinella septempunctata L. and Hippodamia variegata Goeze are the most effective predators upon Aphis gossypii Glov., which is an economically important pest of melon. Understanding their likelihood to engage in reciprocal predation is a key point for conservation of biological control. Here, we aim to investigate, under laboratory conditions, the level of IGP between the two above mentioned aphidophagous species. (2) Fourth-instars of the two species were isolated in petri dishes with combinations of different stages of the heterospecific ladybird and different densities of A. gossypii. The occurrence of IGP events was recorded after six hours. (3) C. septempunctata predated H. variegata at a higher rate than vice versa (70% vs. 43% overall). Higher density of the aphid or older juvenile stage of the IG-prey (22% of fourth instars vs. 74% of eggs and second instars) reduces the likelihood of predation. (4) To our knowledge, IGP between C. septempunctata and H. variegata was investigated for the first time. Results represent a baseline, necessary to predict the likelihood of IGP occurrence in the field.