Variability of the mitochondrial gene for cytochrome oxidase I within the Adalia bipunctata species and within species of ladybird beetles (Coleoptera: Coccinellidae)

Intergeneric, interspecific, and intraspecific genetic variation of the 310-bp 3'-end region of the mitochondrial gene of cytochrome oxidase I (COI) has been assessed in ladybirds (Coleoptera: Coccinnellidae). The phylogenetic distances between eight species of ladybirds have been determined. Mitochondrial DNA (mtDNA) nucleotide sequences have been compared in Adalia bipunctata L. differing in the elytron and pronotum colors that have been sampled from several geographically remote populations. The taxonomic statuses of two morphs from the genus Adalia, A. bipunctata bipunctata and A. bipunctata fasciatopunctata, have been identified.     

Assessment of patch quality by ladybirds: role of larval tracks

Gravid females of the two-spot ladybird, Adalia bipunctata (L.), were deterred from ovipositing when kept in petri dishes that had previously contained conspecific larvae but not conspecific adults, or the larvae of another two species of ladybird, Adalia decempunctata (L.) and Coccinella septempunctata L. The deterrent effect was density dependent and mediated via a chloroform-soluble contact pheromone present in the larval tracks. Similarly, gravid females of C. septempunctata were deterred from ovipositing by conspecific larval tracks and chloroform extracts of these tracks, but not by the tracks or extracts of tracks of A. bipunctata larvae. That is, in ladybirds the larvae produce a species-specific oviposition-deterring pheromone. In the field, the incidence of egg cannibalism in ladybirds increases very rapidly with the density of conspecific eggs or larvae per unit area. Thus, in responding to the species specific oviposition deterring pheromone female ladybirds reduce the risk of their eggs being eaten and spread their offspring more equally between patches. Received: 14 March 1997 / Accepted: 26 August 1997     

Expansion of the geographical distribution of an exotic ladybird beetle, Adalia bipunctata (Coleoptera: Coccinellidae), and its interspecific relationships with native ladybird beetles in Japan

The first record of the exotic ladybird beetle, Adalia bipunctata (Coleoptera; Coccinellidae), in Japan was in 1993 at Osaka Nanko Central Park. Since that time, studies on the life history and geographical distribution of A. bipunctata have been ongoing, and its establishment in the Osaka Nanko area has been confirmed. A. bipunctata is a predacious ladybird beetle and a member of a guild that overlaps in habitat and prey with that of native ladybird beetles such as Harmonia axyridis and Menochilas sexmaculatus. We investigated the distribution of A. bipunctata and its interspecific relationships with native predacious ladybird beetles. In some areas, A. bipunctata was dominant in interspecific relationships with native ladybird beetles. For the first 10 years after A. bipunctata was discovered, it occurred only in the Osaka Nanko area, but the present geographical distribution indicates that it has expanded its range. Though the population density of this species was highest at the area recorded first, and tended to decrease in inverse proportion to the distance from Osaka Nanko Central Park, a satellite occurrence was observed in a remote area. The numbers of aphid and tree species (leaf shelter for aestivation and over-wintering) utilized by A. bipunctata have recently increased. Such increases will cause the rate of distribution of A. bipunctata to accelerate. Interspecific competition between H. axyridis and A. bipunctata, which occurs earlier than H. axyridis, may be avoided by desynchronization of the occurrence seasons, and another common predacious ladybird beetle, Coccinella septempunctata, may escape interspecific competition by habitat segregation.     

Does the satiation hypothesis account for the differences in efficacy of coccidophagous and aphidophagous ladybird beetles in biological control? A test with Adalia bipunctata and Cryptolaemus montrouzieri

Mills (1982) formulated the satiationhypothesis in order to explain why ladybirdsare generally more effective in the biologicalcontrol of coccids than of aphids:aphidophagous ladybirds are supposedly lessefficient because they become more rapidlysatiated than coccidophagous ladybirds. If thisis true, the former should spend less timeeating than the latter. This hypothesis istested in this study using the coccidophagousCryptolaemus montrouzieri Mulsant(Coleoptera: Coccinellidae) and theaphidophagous Adalia bipunctata (L.) (Coleoptera: Coccinellidae). Contrary to Mills'(1982) prediction the coccidophagous ladybirddid not feed continuously and even spent lesstime feeding than the aphidophagous ladybird.Furthermore, the gut capacity of C. montrouzieri is smaller than that of A. bipunctata.     

Geographical Ecocline Polymorphism of the Distribution of Mitochondrial Haplotypes of <Emphasis Type="Italic">Adalia bipunctata</Emphasis> Linnaeus, 1758 (Coleoptera: Coccinellidae) of Norway

Adalia bipunctata Linnaeus, 1758 is a classical object for studying polymorphism of natural populations. Application of genetic markers significantly increased the possibilities of analysis of natural populations of ladybirds and opened the possibility of studying ecological adaptations at the genetic level. We analyze the geographical distribution of the mitochondrial haplotypes of A. bipunctata, determined on the basis of the variability of the mitochondrial cox1 gene in the natural populations of Norway. Two new mitochondrial haplotypes of A. bipunctata are described. We observe a geographical cline in the haplotypes frequencies from south to north. The main haplotype H1 of the central Europe is gradually replaced in the north by the haplotype H7. A possible reason for the observed geographical cline is a selective advantage of beetles with haplotype H7 in areas with a northern coastal climate.     

Effects of competition,cannibalism and intra‐guild predation on larval development of the European coccinellid Adalia bipunctata and the invasive species Harmonia axyridis

Abstract 1. Resource competition and intra‐guild predation (IGP) are important determinants of the structure of aphidophagous guilds. The likelihood and outcome of IGP is influenced by the density of extra‐guild prey and the characteristics of the species involved. 2. The nature of intra‐guild interactions between alien and indigenous coccinellids often determines the success of coccinellid invasions, as exemplified by Adalia bipunctata in Japan and Harmonia axyridis in North America and Europe. 3. Harmonia axyridis has negatively impinged on indigenous species in its introduced range, and its recent arrival in Britain poses a threat for members of native aphidophagous guilds. IGP and inter‐specific competition between H. axyridis and British coccinellids are predicted to occur. However, the results of such interactions have been little studied. 4. Here we investigate the effects of different diets, designed to mimic possible conditions in the wild, on the survival, development, and adult size of H. axyridis and A. bipunctata. Results demonstrate a skew in the consequences of IGP between the two species: the supplementation of a limited aphid diet with non‐conspecific eggs leads to a significant advantage for H. axyridis in respect of all parameters assessed, but gives no benefit to A. bipunctata. 5. We conclude that IGP of A. bipunctata by H. axyridis will contribute to the spread and increase of H. axyridis in Britain. 6. We further conclude that the skewed nature of IGP between A. bipunctata and H. axyridis at least in part explains the limited spread of A. bipunctata in Japan.     

A study of nonrandom mating in a British population of the two-spot ladybird with a high frequency of the melanic morph

In some studies of the two-spot ladybird (Adalia bipunctata), melanic males have been found in excess over the typical morph in matings. Data suggest that a genetic female mating preference is responsible. The mating advantage of melanic males may be important in maintaining a polymorphism between melanic and typical ladybirds in many populations in the United Kingdom (U.K.). It has been reported that preference frequency varies linearly with melanic frequency throughout most of the U.K. One particular population ofAdalia bipunctata near Aberdare, South Wales, is noted for its high frequency of melanic individuals. It has been suggested that local environmental factors account for the high melanic frequency in this population. It is also possible, however, that a female mating preference may be at least partly responsible for the high frequency of melanics (as has been proposed for the rest of the U.K.). In this study, experiments have been performed to determine the level of female mating preference in the Aberdare population. No evidence was found for any mating advantage to melanic males. There was inconsistent and unexpected evidence that melanic females were overrepresented in matings, but the cause for this was unclear. Female mating preference does not appear, therefore, to be responsible for the high melanic frequency in the population ofAdalia bipunctata near Aberdare. There is not a simple association between mating preference and melanic frequency in U. K. populations of the two-spot ladybird.     

Egg composition and reproductive investment in aphidophagous ladybird beetles (Coccinellidae: Coccinellini): egg development and interspecific variation

Abstract Most studies of insect reproductive allocation concentrate on propagule size and number and very few consider egg composition, which is likely to be equally important. In the present study, data are provided on changes in egg lipid, glycogen, free carbohydrate and protein during embryonic development of the aphidophagous ladybird Adalia bipunctata (L.) and the compositions of A. bipunctata, Adalia decempunctata and Anisosticta novemdecimpunctata eggs are compared. In A. bipunctata, egg mass, lipid and glycogen decline strongly during development and egg protein declines more weakly. Free carbohydrate declines early in egg development and increases at egg hatching. Lipid is energetically the most important developmental fuel, although approximately half of the initial egg lipid remains in the neonate larva. Across the three species, energy per unit egg mass is lowest in the least specialized species, A. bipunctata, which also has the largest eggs, and is highest in the most specialized, An. novemdecimpunctata, which has the smallest eggs. Two possible explanations for the observed pattern are discussed: (i) species laying smaller eggs may incur higher developmental costs per unit mass than species laying larger eggs and (ii) more specialized species, which reproduce at lower aphid densities, may provision neonate larvae better to facilitate location and capture of aphids.     

Estimation of mortality by entomophages on exotic Harmonia axyridis versus native Adalia bipunctata in semi-field conditions in northern Italy

A semi-field experiment was carried out in two peach orchards in northern Italy to assess mortality due to predators and parasitoids on the exotic coccinellid Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in comparison with the native coccinellid Adalia bipunctata L. (Coleoptera: Coccinellidae). The experiments were conducted in cages to avoid the possible escape of the exotic ladybird (not yet established in Italy). Two kinds of cage experiments were included: ‘exclusion cages’ (access by walking predators impeded) and ‘free cages’ (walking predators free to enter). The cages, containing all the stages of the two ladybird species, were placed in two localities and left for 24 h. All ladybird stages used for the semi-field experiments came from a laboratory rearing. The eggs of H. axyridis experienced less mortality than those of A. bipunctata. The ant workers were the most frequent predators in ‘free cages’ but A. bipunctata cannibalism on eggs was also detected. Larvae of both coccinellid species were predated equally but larval predation of L1 and L2 was higher in comparison to predation of L3 and L4. Pupae and adults of both exotic and native ladybirds were never attacked by predators. Predation on younger larval stages was higher in the ‘free cages’ in comparison with ‘exclusion cages’. No ladybird parasitisation was observed. The ‘free cage’ technique seems to provide a standardised and realistic estimation of predation impact but more studies are needed to evaluate ladybird parasitisation in semi-field conditions.     

Laboratory study of cannibalism and interspecific predation in ladybirds

Abstract.

• 1 In the absence of aphids, adult females of Adalia bipunctata (L.) showed a greater reluctance to eat eggs than males.
• 2 Eggs and young larvae were more vulnerable to cannibalism than older larvae and starved larvae were more vulnerable than well-fed larvae.
• 3 Both egg and larval cannibalism is inversely related to the abundance of aphids.
• 4 Eggs are a better food, in terms of larval growth and survival, than aphids.
• 5 In the absence of aphids interspecific predation occurred, but not equally, between the coccinellids A.bipunctata, A.decempunctata (L.), Coccinella septempunctata L. and C.undecempunctata L.
• 6 Larvae and adults of A. bipunctata and C.septempunctata were reluctant to eat conspecific eggs painted with a water extract of the other species' eggs and larvae of C. septempunctata were more likely to die after eating a few eggs of A.bipunctata than vice versa.
• 7 These results indicate that cannibalism occurs mainly when aphid prey is scarce and is adaptive in that it improves the chances of survival, and coccinellids, to varying degrees, are defended against interspecific predation.

     

Tritrophic Interactions Between Transgenic Potato Expressing Snowdrop Lectin (GNA), an Aphid Pest (Peach–Potato Aphid; Myzus Persicae (Sulz.) and a Beneficial Predator (2-Spot Ladybird; Adalia Bipunctata L.)

Tritrophic interactions between transgenic potato expressing the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA), an aphid pest, Myzus persicae (Sulz.), and a beneficial predator, the 2-spot ladybird (Adalia bipunctata L.) were investigated. Clonal plants expressing GNA at 0.1–0.2% total soluble protein in leaves were used. No significant effects on development and survival of ladybird larvae fed on aphids from these transgenic plants were observed, with larval survival in the experimental group being 90% compared to 89% for controls. There were also no effects on subsequent female or male longevity. Female fecundity was also investigated. Although no significant differences (p > 0.05) were observed in egg production between control and experimental groups, a 10%, reduction (p < 0.01) in egg viability (determined by % hatch) occurred in ladybirds fed aphids reared on transgenic plants. Additional studies were carried out using aphids fed on artificial diet containing GNA, to deliver quantified levels of the protein to ladybird adults. GNA had no deleterious effects upon adult longevity, but resulted in a consistent trend for improved fecundity. Egg production was increased by up to 70% and egg viability also increased significantly. The results suggest that GNA is not deleterious to ladybirds. Results from these studies highlight the need to discriminate between direct and indirect effects when studying tritrophic interactions between plants/pests/natural enemies. Furthermore, it emphasises the importance of demonstrating cause and effect.     

Distribution of cytoplasmically inherited bacteria Spiroplasma causing female bias in the Eurasian populations of Adalia bipunctata

Two-spot ladybirds Adalia bipunctata were collected from the populations of Western and Eastern Europe and Central Asia. The agent killing males at the early embryonic stage in these populations was identified as bacteria of the genus Spiroplasma. Bacteria found in A. bipunctata proved to be phylogenetically related to Spiroplasma ixodetis (typical line Y-32) found in tick Ixodes pacificus but not to Spiroplasma causing the death of male embryos in Drosophila.     

Long term changes in the communities of native ladybirds in Northern Italy: impact of the invasive species <Emphasis Type="Italic">Harmonia axyridis</Emphasis> (Pallas)

Since the mid-2000s, the exotic coccinellid species Harmonia axyridis (Pallas) has established itself in Northern Italy, raising serious concerns about possible repercussions on native ladybirds. In this study we compared the ladybird assemblages in 2015–2016 with those sampled in 1995–1996, before the arrival of H. axyridis. Surveys were carried out in the same sites and with the same methods for both periods. Aphidophagous ladybirds were sampled in hedgerows and herbaceous habitats at field margins by mechanical knockdown and sweeping net, respectively. The changes in the structure of ladybird communities after the arrival of H. axyridis were significantly different between arboreal and herbaceous habitats. Harmonia axyridis is currently the dominant species in shrubs and trees, and all the native ladybird species taken together account for only approximately one third of the total individuals sampled in 2015–2016. On the other hand, the relative abundance of the exotic species in herbaceous habitats was low, and it has had a negligible relevance on the structure of ladybird communities on grasslands. Among native species, Adalia bipunctata (L.), whose ecological niche largely overlaps with that of H. axyridis, suffered the largest decline between 1995–1996 and 2015–2016. The co-occurrence of H. axyridis invasion and the regression of A. bipunctata suggest a direct impact of the exotic species, because no other major modifications occurred in the studied areas between sampling periods.     

Decline in native ladybirds in response to the arrival of Harmonia axyridis: early evidence from England

1. Harmonia axyridis (Coleoptera: Coccinellidae) is an invasive non‐native ladybird in Europe, where it was introduced as a biological control agent of aphids and coccids. 2. This study assesses changes to ladybird species assemblages, in arboreal habitats, over a 3‐year period encompassing the invasion phase of H. axyridis in eastern England. The effects of H. axyridis and other factors (weather and prey availability) on native ladybirds are assessed. 3. Harmonia axyridis increased from 0.1% to 40% of total ladybirds sampled, whilst native aphidophagous species declined from 84% to 41% of total ladybirds. The actual number of native aphidophagous ladybirds per survey decreased from a mean of 19.7 in year 1, to 10.2 in year 3. 4. Three ladybird species in particular experienced declines: Adalia bipunctata, Coccinella septempunctata, and Propylea quattuordecimpunctata. Harmonia axyridis was the most abundant species by the end of the study. 5. The decline in native aphidophagous ladybirds could be attributed to competition for prey and intraguild predation of eggs, larvae, and pupae by H. axyridis. Physiological and behavioural traits of H. axyridis are likely to confer an advantage over native ladybird species.     

The effects of different aphid foods on Adalia bipunctata L. and Coccinella 7-punctata L

The influence of different aphid foods on larval development and adult fecundity of Adalia bipunctata L. and Coccinella 7-punctata L. was investigated. Certain aphids such as Myzus persicae Sulz. and Acyrthosiphon pisum Harris were suitable for both species. Megoura viciae Buckt., was toxic to A. bipunctata but not to C. 7-punctata. Aphis fabae Scop., although a common natural prey of A. bipunctata, slowed larval development, partly because it was nutritionally unsuitable. Fecundity of adults fed on A. fabae was also reduced to less than half. Larvae and adults of C. 7-punctata developed and reproduced as well on A. fabae as on other suitable aphids. C. 7-punctata caught aphids more quickly than A. bipunctata. Larvae of both species given relatively unsuitable prey fed more slowly than normal and consumed less of each aphid. The toxic aphid M. viciae was rejected by A. bipunctata after a short period of feeding.     

Isolation and characterization of highly polymorphic microsatellite loci in the 2‐Spot Ladybird,Adalia bipunctata

Contrary to theoretical predictions, female 2‐spot ladybirds (Adalia bipunctata) mate many more times than necessary to maintain high fertilisation success and may gain through the acquisition of material or genetic benefits. In order to investigate this mating system in detail, microsatellite markers have been isolated using a modified enrichment technique. Thirty‐nine loci were successfully amplified by polymerase chain reaction (PCR), of which only two were monomorphic. Detailed characterization of ten loci revealed very high levels of polymorphism. These markers are likely to be invaluable tools with which to study population genetics and patterns of paternity in this species.     

Natural flightless morphs of the ladybird beetle Adalia bipunctata improve biological control of aphids on single plants

The challenge of using ladybird beetles for biological control of insect pests such as aphids is that the adult beetles tend to fly away from the host plants. Therefore, flightless ladybirds might improve biocontrol. There are several artificial ways to obtain flightless beetles, but it may be preferable to use natural variation in flight ability. We investigated, for the first time, biocontrol by inundative augmentation of natural flightless morphs of the ladybird beetle Adalia bipunctata. Microcosm experiments using single leaves with one of three species of aphid revealed no differences in consumption behavior between flightless and winged beetles. Monitoring for 48 h of single, caged pepper plants infested with aphids of Myzus persicae nicotianae or Aulacorthum solani showed that flightless beetles had a longer residence time on the plants than winged beetles. This only translated into significantly better biocontrol of M. persicae. Despite their difference in residence time, both beetle morphs reduced the population growth of A. solani. This is probably explained by the tendency of A. solani to drop from the plant upon disturbance, and we predict that flightless beetles may outperform winged ones in the long term. Overall, our results provide a proof of principle that natural flightless A. bipunctata can improve biocontrol of aphids by ladybird beetles. However, we recognize that the effect of biocontrol will vary with the species of aphid used and that further examination in long term and large scale experiments is required.     

Presence of haemocyte-like cells in coccinellid reflex blood

Abstract.  Contrary to current assumptions, the reflex blood of two-spot ladybirds, Adalia bipunctata , and seven-spot ladybirds, Coccinella septempunctata , contains haemocyte-like cells. Furthermore, DNA can be extracted and amplified from coccinellid reflex blood, confirming the presence of haemocyte-like cells and demonstrating a nondestructive method of DNA extraction.     

Ladybird egg cluster size: relationships between species, oviposition substrate and cannibalism

The success or not of ladybirds as biological control agents is dependent on both their foraging behaviour and their individual survival rates. The former is a function of the habitats they utilise; the latter, a consequence of their reproductive strategy. Egg clustering was investigated in two ladybird species, Aphidecta obliterata, a conifer specialist, and Adalia bipunctata, an arboreal woodland generalist. The effect of oviposition substrate (filter papers vs. spruce needles) on clutch size and oviposition preference was also tested. Adalia bipunctata laid significantly more eggs than A. obliterata. The size of egg clusters laid by the two coccinellids varied between species and substrate types. Adalia bipunctata laid larger egg clusters than A. obliterata, with batches reaching a maximum size of 32 eggs on spruce and 41 eggs on paper, while those of A. obliterata contained a maximum of 5 eggs on spruce and 9 eggs on paper. Of the clusters laid by A. obliterata, 18.6% of those on paper and 21.4% of those on spruce contained only a single egg, whereas a minimum of two eggs per cluster were laid by A. bipunctata. Smaller clusters were laid on the spruce cuttings by both species when compared with those laid on the filter paper, but A. obliterata laid significantly more eggs on spruce than on the filter paper (77% vs. 23%), whilst A. bipunctata laid significantly more eggs on the filter paper (91%). It is suggested that coccinellid eggs are more likely to be washed off spruce needles than broad leaves and that, by laying smaller egg clusters on spruce, A. obliterata reduces this risk. Adalia bipunctata usually lays its eggs on the underside of broad leaved trees and thus does not face this risk and thus can lay larger egg clusters. No differences in cannibalism rates were found between the two species. These findings have implications for the use of ladybirds as biological control agents in spruce forests.     

Effect of intraguild predation on the survival and development of three species of aphidophagous ladybirds: consequences for invasive species

Abstract 1 Survival and development of hatchling larvae of three aphidophagous ladybirds (Coleoptera: Coccinellidae), Harmonia axyridis Pallas, Coccinella septempunctata brucki Mulsant and Adalia bipunctata Linnaeus, when fed their own and the other species eggs were recorded. 2 In all three species, the larvae survived when fed conspecific eggs. 3 The percentage of larvae of H. axyridis that survived decreased to 35% and 85% when fed eggs of A. bipunctata and C. s. brucki, respectively. All the larvae of A. bipunctata and C. s. brucki died after eating eggs of H. axyridis. None of the larvae of C. s. brucki died after eating eggs of A. bipunctata, whereas 46% of those of A. bipunctata died after eating eggs of C. s. brucki. 4 In general, larvae were reluctant to eat the eggs of other species. However, larvae of C. s. brucki showed less reluctance than H. axyridis to eat the eggs of A. bipunctata. 5 The consequence of this for invasive species of ladybird is discussed.