Cold tolerance of the harlequin ladybird Harmonia axyridis in Europe

As an essential aspect of its invasive character in Europe, this study examined the cold hardiness of the harlequin ladybird Harmonia axyridis. This was done for field-collected populations in Belgium overwintering either in an unheated indoor or an outdoor hibernaculum. The supercooling point, lower lethal temperature and lower lethal time at 0 and −5 °C were determined. Possible seasonal changes were taken into account by monitoring the populations during each winter month. The supercooling point and lower lethal temperature remained relatively constant for the overwintering populations in the outdoor hibernaculum, ranging from −17.5 to −16.5 °C and −17.1 to −16.3 °C, respectively. In contrast, the supercooling point and lower lethal temperature of the population overwintering indoors clearly increased as the winter progressed, from −18.5 to −13.2 °C and −16.7 to −14.1 °C, respectively. A proportion of the individuals overwintering indoors could thus encounter problems surviving the winter due to premature activation at times when food is not available. The lower lethal time of field populations at 0 and −5 °C varied from 18 to 24 weeks and from 12 to 22 weeks, respectively. Morph type and sex had no influence on the cold hardiness of the overwintering adults. In addition, all cold tolerance parameters differed greatly between the laboratory population and field populations, implying that cold tolerance research based solely on laboratory populations may not be representative of field situations. We conclude from this study that the strong cold hardiness of H. axyridis in Europe may enable the species to establish in large parts of the continent.     

Entomopathogenic fungi recorded from the harlequin ladybird, Harmonia axyridis

Entomopathogenic fungi were recorded from field samples of the harlequin ladybird Harmonia axyridis, an invasive coccinellid that has recently arrived in Denmark. Larvae, pupae and adults were found to be infected by Isaria farinosa, Beauveria bassiana and species of Lecanicillium. This is the first record of entomopathogenic fungi infecting larvae and pupae. Winter mortality due to fungal infection reached 17.9% in adults collected at one location. The larval stage was most susceptible to fungal infection, as confirmed through bioassay with I. farinosa.     

Predicting the potential geographical distribution of the harlequin ladybird, Harmonia axyridis, using the CLIMEX model

Harmonia axyridis (Pallas, 1773) (Coleoptera: Coccinellidae) is a ladybird beetle native to temperate and subtropical parts of Asia. Since 1916 populations of this species have been introduced throughout the world, either deliberately, or by accident through international transport. Harmonia axyridis was originally released as a classical biological control agent of aphid and coccid pests in orchards and forests, but since 1994 it is also available as a commercial product for augmentative control in field and greenhouse crops. It is a very voracious and effective natural enemy of aphids, psyllids and coccids in various agricultural and horticultural habitats and forests. During the past 20 years, however, it has successfully invaded non-target habitats in North America (since 1988), Europe (1999) and South America (2001) respectively in a short period of time, attacking a wide range of non-pest species in different insect orders. Becoming part of the agricultural commercial pathway, it is prone to being introduced into large areas across the world by accident. We use the CLIMEX programme (v2) to predict the potential geographical distribution of H. axyridis by means of matching the climate of its region of origin with other regions in the world and taking in account biological characteristics of the species. Establishment and spread seem likely in many regions across the world, including those areas which H. axyridis has already invaded (temperate Europe, North America). Based on the CLIMEX prediction a large part of Mediterranean Europe, South America, Africa, Australia and New Zealand seem highly suitable for long-term survival of H. axyridis as well. In addition we evaluate CLIMEX as a strategic tool for estimating establishment potential as part of an environmental risk assessment procedure for biological control agents we discuss biological and ecological aspects necessary to fine-tune its establishment and spread in areas after it has been introduced.     

Phenotypic variation in invasive and biocontrol populations of the harlequin ladybird, Harmonia axyridis

Despite numerous releases for biological control purposes during more than 20 years in Europe, Harmonia axyridis failed to become established until the beginning of the 21st century. Its status as invasive alien species is now widely recognised. Theory suggests that invasive populations should evolve toward greater phenotypic plasticity because they encounter differing environments during the invasion process. On the contrary, populations used for biological control have been maintained under artificial rearing conditions for many generations; they are hence expected to become specialised on a narrow range of environments and show lower phenotypic plasticity. Here we compared phenotypic traits and the extent of adaptive phenotypic plasticity in two invasive populations and two populations commercialized for biological control by (i) measuring six phenotypic traits related to fitness (eggs hatching rate, larval survival rate, development time, sex ratio, fecundity over 6 weeks and survival time of starving adults) at three temperatures (18, 24 and 30°C), (ii) recording the survival rate and quiescence aggregation behaviour when exposed to low temperatures (5, 10 and 15°C), and (iii) studying the cannibalistic behaviour of populations in the absence of food. Invasive and biocontrol populations displayed significantly different responses to temperature variation for a composite fitness index computed from the traits measured at 18, 24 and 30°C, but not for any of those traits considered independently. The plasticity measured on the same fitness index was higher in the two invasive populations, but this difference was not statistically significant. On the other hand, invasive populations displayed significantly higher survival and higher phenotypic plasticity when entering into quiescence at low temperatures. In addition, one invasive population displayed a singular cannibalistic behaviour. Our results hence only partly support the expectation of increased adaptive phenotypic plasticity of European invasive populations of H. axyridis, and stress the importance of the choice of the environmental parameters to be manipulated for assessing phenotypic plasticity variation among populations.     

Mechanisms of prey finding in the aphidophagous ladybird beetle,Harmonia axyridis [Coleoptera: Coccinellidae]

The mechanism of prey finding in adultHarmonia axyridis was investigated by presenting beetles with small gauze or polyethylene bags containing either aphidinfested leaves or healthy leaves alone together with empty control bags. Beetles were attracted to bags by olfactory and visual cues suggesting the presence of aphids, including the odor of aphid-infested leaves, the odor of aphids, the odor of healthy leaves, and the sight of leaves. Particularly effective in prolonging beetles's stay on bags visited was the odor of aphid-infested leaves. It is suggested that adults ofH. axyridis use olfactory and visual cues to detect prey before actual contact occurs. Use of such cues seems more advantageous than random searching, as has been reported in many previous studies of coccinellid foraging.

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Résumé Le mécanisme de découverte de la proie chez l'adulte d'Harmonia axyridis est étudié en donnant aux Coccinelles de petits sacs en gaze ou en polyéthylène contenant soit des feuilles infestées de pucerons, soit des feuilles saines seules, avec des sacs vides comme témoins. Les coccinelles sont attirées vers les sacs par des indicateurs olfactifs ou visuels suggérant la présence de pucerons, incluant l'odeur des feuilles infestées de pucerons, l'odeur des pucerons, l'odeur des feuilles saines et la vue des feuilles. L'odeur des feuilles infestées de pucerons est particulièrement efficace en prolongeant le séjour des Coccinelles sur le sac visité. Il est suggéré que les adultes d'H. axyridis utilisent des indicateurs olfactifs et visuels pour détecter leur proie avant que s'établisse le véritable contact. L'emploi de tels indicateurs semble plus avantageux que la recherche au hasard, comme cela a été indiqué dans de nombreuses études antérieures sur la quête alimentaire des Coccinelles.

     

Antimicrobial defence and persistent infection in insects revisited

Insects show long-lasting antimicrobial immune responses that follow the initial fast-acting cellular processes. These immune responses are discussed to provide a form of phrophylaxis and/or to serve as a safety measure against persisting infections. The duration and components of such long-lasting responses have rarely been studied in detail, a necessary prerequisite to understand their adaptive value. Here, we present a 21 day proteomic time course of the mealworm beetle Tenebrio molitor immune-challenged with heat-killed Staphylococcus aureus. The most upregulated peptides are antimicrobial peptides (AMPs), many of which are still highly abundant 21 days after infection. The identified AMPs included toll and imd-mediated AMPs, a significant number of which have no known function against S. aureus or other Gram-positive bacteria. The proteome reflects the selective arena for bacterial infections. The results also corroborate the notion of synergistic interactions in vivo that are difficult to model in vitro.This article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’.     

A natural flightless mutation in the ladybird, Harmonia axyridis

A homozygous flightless strain, obtained by selection from a laboratory population of Harmonia axyridis Pallas (Coleoptera, Coccinellidae), has been reared under controlled conditions since 1982 after sampling in China. The adults of this flightless strain have normal elytra and wings but drop almost vertically when they attempt to fly. The origin of this mutation, whether laboratory or natural, is discussed. As the mutation does not affect the fitness of the beetles, this flightless strain can be used in biological control. Releasing flightless adults rather than larvae should provide the potential for more continuous control of aphids by both larvae and adults.     

Antennal sensory organs and glands of the harlequin ladybird,Harmonia axyridis

Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is a sub‐cosmopolitan species. Native to Asia, it has been released during the 20th century for classical and augmentative biological control of several herbivorous insects, mostly aphids and coccids. Despite its recognized positive impact on biological control, H. axyridis is now considered among the most dangerous invasive species in Europe and in most places where it has established. This is mostly due to its ability to reduce the populations of native predatory species of the same trophic guild. When exploring a new area, H. axyridis adults use semiochemical cues to acquire information about the habitat. Presumably, these cues are perceived by the sensilla located on the antennae. Surprisingly, in spite of the huge literature existing on H. axyridis, the antennal sensory organs have been poorly characterized. Here, we used scanning and transmission electron microscopy (SEM, TEM) techniques to study H. axyridis antennae, with focus on the various types of sensilla and their distribution in male and female individuals. The presence of various classes of antennal sensilla belonging to the main types described in insects (chemoreceptors, mechanoreceptors, and thermo‐hygroreceptors) was highlighted, as well as the widespread presence of antennal glands. The investigations showed some peculiar characteristics not known in Coccinellidae, such as the concentration of sensory structures at the level of the distal part of the apical antennomere and the discovery of antennal glands associated with it. No sexual dimorphism was revealed, neither for the general structure of the antenna (similar number of antennomeres and presence of modifications), nor for the total length and width of the antenna, the relative size of the antennomeres, the types of antennal sensilla, of their distribution and abundance. The potential relevance of these sensory structures and antennal glands, reported for the first time in Coccinellidae, is discussed in the context of intra‐ and interspecific communication.     

PvD1 defensin,a plant antimicrobial peptide with inhibitory activity against Leishmania amazonensis

Plant defensins are small cysteine-rich peptides and exhibit antimicrobial activity against a variety of both plant and human pathogens. Despite the broad inhibitory activity that plant defensins exhibit against different micro-organisms, little is known about their activity against protozoa. In a previous study, we isolated a plant defensin named PvD₁ from Phaseolus vulgaris (cv. Pérola) seeds, which was seen to be deleterious against different yeast cells and filamentous fungi. It exerted its effects by causing an increase in the endogenous production of ROS (reactive oxygen species) and NO (nitric oxide), plasma membrane permeabilization and the inhibition of medium acidification. In the present study, we investigated whether PvD₁ could act against the protozoan Leishmania amazonensis. Our results show that, besides inhibiting the proliferation of L. amazonensis promastigotes, the PvD₁ defensin was able to cause cytoplasmic fragmentation, formation of multiple cytoplasmic vacuoles and membrane permeabilization in the cells of this organism. Furthermore, we show, for the first time, that PvD₁ defensin was located within the L. amazonensis cells, suggesting the existence of a possible intracellular target.     

Interference competition between ladybird beetle adults (Coleoptera: Coccinellidae): effects on growth and reproductive capacity

Cage experiments were used to determine the minimum number of Aphis fabae Scopoli that females of the Azorean native ladybird beetle Coccinella undecimpunctata L. require per day to achieve maximum fecundity and to assess the effects of interference competition from conspecific and heterospecific [Harmonia axyridis (Pallas)] adult ladybirds on its growth and reproduction. The number of A. fabae at which females of C. undecimpunctata ate most aphids, achieved maximum reproduction and maintained their body weight, was 200. The presence of H. axyridis adults, unlike the presence of C. undecimpunctata, significantly affected the reproductive numerical response of C. undecimpunctata even though the resource was not in short supply. The results suggest that interference competition with heterospecifics may adversely affect the reproductive capacity of C. undecimpunctata. Based on previous results and those reported here, we suggest that the introduction of H. axyridis could endanger the population abundance and/or result in the competitive displacement of C. undecimpunctata from the Azorean ecosystem.     

Ontogeny of protein concentration,haemocyte concentration and antimicrobial activity against Escherichia coli in haemolymph of the invasive harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae)

The harlequin ladybird is considered to be one of the most successful invasive insect species. Among other traits, its invasive success is considered to be caused by a powerful immune system. In the present study, we investigate the ontogenetic profile of protein concentration, concentration of circulating haemocytes and constitutive antimicrobial activity against Escherichia coli in Harmonia axyridis haemolymph during late larval development and early adult life. Protein concentration increases during the first 32 days of adult life from 45 to 100 mg per mL of haemolymph and reaches intermediate values during larval stages. The concentration of circulating haemocytes is very low (5000 haemocytes per μL of haemolymph) in late larval stages and increases strongly during first 8 days of adult life to values of approximately 30 000 haemocytes per μL of haemolymph. The killing efficiency of haemolymph against E. coli is lowest in larval stages, rapidly increases in the prepupal stage and then steadily grows during the whole period of adult life. There are no significant effects of sex on any of the investigated physiological or immune parameters. In general, the patterns observed for H. axyridis contrast with many results that are reported for other insects (e.g. bees, fruit flies, crickets or mosquitoes). One possible explanation is the contrasting life history of H. axyridis, with a fast preimaginal development and a long adult lifespan being linked to a long reproductive period. Substantial variation in physiological and immune parameters during ontogeny also has important methodological implications because individuals of exactly the same stage/age have to be employed for comparative studies.     

Exposure to sexual signals during rearing increases immune defence in adult field crickets

Increased investment in immunity is expected to be beneficial under crowded conditions because of the greater risk of pathogen and parasite transmission, but the evolution of this facultative response relies on the ability to accurately assess social cues in the environment and adjust immune defences accordingly. Because of their highly conspicuous nature, long-range sexual signals are prime candidates to be used in evaluating the social conditions likely to be experienced upon adulthood in continuously breeding species; however, their role in mediating immune responses is unknown. We tested whether exposure to acoustic sexual signals in the field cricket Teleogryllus oceanicus affects immunity by manipulating male juvenile experience of acoustic signals, and measuring the effect on adult immunity. Adult males exposed to song during rearing showed stronger immune responses than males reared in silence: they were better able to encapsulate artificial nylon implants and showed higher levels of antimicrobial lysozyme-like activity in their haemolymph. Experience of sexual signals thus translates into increased immunity, which suggests that such signals may play a role in conveying information about population demography and shaping density-dependent responses in unintended receivers.     

A new ladybird spider from Hungary (Araneae,Eresidae)

According to the most recent taxonomic literature, three species of the genus Eresus are known in Central Europe, Eresus kollari, Eresus sandaliatus and Eresus moravicus. We recognized a fourth distinctive species from Hungary, which is described as Eresus hermani sp. n. Eresus hermani has an early spring copulation period, females have a light grey (grizzled) cephalothorax due to a heavy cover of lightly colored setae, and an epigyne with large flat areas posterior to the epigynal pit, while males are distinguished by a broad and blunt terminal tooth of the conductor. An updated and modified comparative table of Řezáč et al. (2008) to include all four Central European Eresus species, and a simple key to the species group’s species are given. Habitus, epigyne, vulva and conductor of Eresus kollari, Eresus moravicus and Eresus sandaliatus are also illustrated. An annotated list of papers illustrating Eresus hermani due to misidentifications is presented.     

In vitro and in vivo antimalarial activity and cytotoxicity of extracts,fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.     

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation.     

Rourinoside and rouremin, antimalarial constituents from Rourea minor

Bioassay-directed fractionation of the antimalarial active CHCl(3) extract of the dried stems of Rourea minor (Gaertn.) Aubl. (Connaraceae) liana led to isolation of two glycosides, rourinoside (1) and rouremin (2), as well as five known compounds, 1-(26-hydroxyhexacosanoyl)-glycerol (3), 1-O-beta-D-glucopyranosyl-(2S,3R,4E-8Z)-2-N-(2'-hydroxypalmitoyl)-octadecasphinga-4,8-dienine, 9S,12S,13S-trihydroxy-10E-octadecenoic acid, dihydrovomifoliol-9-beta-D-glucopyranoside, and beta-sitosterol glucoside. Compounds 1-3 showed weak in vitro activities against Plasmodium falciparum. Their structures and stereochemistry were elucidated by spectroscopic methods and selected enzyme hydrolysis.     

Condition-dependent ejaculate size and composition in a ladybird beetle

Sexually selected male ejaculate traits are expected to depend on the resource state of males. Theory predicts that males in good condition will produce larger ejaculates, but that ejaculate composition will depend on the relative production costs of ejaculate components and the risk of sperm competition experienced by low- and high-condition males. Under some conditions, when low condition leads to poorer performance in sperm competition, males in low condition may produce ejaculates with higher sperm content relative to their total ejaculate and may even transfer more sperm than high-condition males in an absolute sense. Previous studies in insects have shown that males in good condition transfer larger ejaculates or more sperm, but it has not been clear whether increased sperm content represents a shift in allocation or simply a larger ejaculate, and thus the condition dependence of ejaculate composition has been largely untested. We examined condition dependence in ejaculate by manipulating adult male condition in a ladybird beetle (Adalia bipunctata) in which males transfer three distinct ejaculate components during mating: sperm, non-sperm ejaculate retained within the female reproductive tract, and a spermatophore capsule that females eject and ingest following mating. We found that high condition males indeed transferred larger ejaculates, potentially achieved by an increased rate of ejaculate transfer, and allocated less to sperm compared with low-condition males. Low-condition males transferred ejaculates with absolutely and proportionally more sperm. This study provides the first experimental evidence for a condition-dependent shift in ejaculate composition.     

Grifolin, a potential antitumor natural product from the mushroom Albatrellus confluens, inhibits tumor cell growth by inducing apoptosis in vitro

Grifolin is a natural biologically active substance isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens. Here, for the first time, we describe a novel activity of grifolin, namely its ability to inhibit the growth of tumor cells by the induction of apoptosis. Grifolin strongly inhibited the growth of tumor cell lines: CNE1, HeLa, MCF7, SW480, K562, Raji and B95-8. Analysis of acridine orange (AO)/ethidium bromide (EB) staining and flow cytometry showed that grifolin possessed apoptosis induction activity to CNE1, HeLa, MCF7 and SW480. Furthermore, the cytochrome c release from mitochondria was detected by confocal microscopy in CNE1 cells after a 12h treatment with grifolin. The increase of caspase-8, 9, 3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by grifolin, and the underexpression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Owing to the combination of the significant antitumor activity by inducing apoptosis and natural abundance of the compound, grifolin holds the promise of being an interesting antitumor agent that deserves further laboratory and in vivo exploration.     

Negative dietary effects of Colorado potato beetle eggs for the larvae of native and introduced ladybird beetles

Aphidophagous ladybird beetles (Coleoptera: Coccinellidae) are attracted to and feed heavily on aphids, but many species will also feed opportunistically on other prey that they encounter. In potatoes (Solanum tuberosum L.) in Washington State, USA, coccinellids feed on both green peach aphids (“GPA,” Myzus persicae Sulzer) and eggs of the Colorado potato beetle (“CPB,” Leptinotarsa decemlineata Say). The guild of aphidophagous ladybirds includes two native species, Hippodamia convergens Guérin-Méneville and Coccinella transversoguttata Brown. Recently, an introduced species, Coccinella septempunctata L., has invaded and apparently displaced its native congener. A second exotic, Harmonia axyridis Pallas, has colonized the area and is becoming more abundant. We compared larval development of each species on a monotypic diet of GPA, a monotypic diet of CPB eggs, or a mixed diet of both GPA and CPB eggs. Our goal was to answer two questions: (1) do larvae of the four ladybird species benefit from including CPB eggs in their diet and (2) do the four ladybird species differ in their ability to utilize CPB eggs as prey? No larva of any species completed development on a pure diet of CPB eggs, and survivorship was highest for all species when they fed on a pure diet of GPA. One native species, H. convergens, and one exotic species, H. axyridis, exhibited significantly lower survivorship on a mixed diet of both CPB eggs and GPA, compared to a pure GPA diet; H. axyridis also took longer to develop from egg to adult when both prey were provided. Survivorship of the two Coccinella spp. was not altered by the inclusion of CPB eggs with GPA, although CPB eggs lengthened the development time of C. transversoguttata. Adult size was not consistently affected by diet for any of the coccinellids. Overall, no ladybird species benefited from the inclusion of potato beetle eggs in its diet. The two Coccinella species responded similarly to the inclusion of CPB eggs, and so we would not expect any difference in the success of coccinellid larval development in potato fields following the replacement of C. transversoguttata by C. septempunctata. Hippodamia convergens and H. axyridis, the two species whose survivorship was depressed by combining CPB egg and aphid prey, were also the two species that consumed the greatest number of CPB eggs during successful larval development. A comparison of total egg consumption by each species cohort suggested that displacement of the other species by H. axyridis would not alter CPB biological control, because the higher per capita feeding rate by H. axyridis larvae compensated for individuals’ greater mortality risk on a diet including CPB eggs.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.