Global genetic diversity,lineage distribution,and Wolbachia infection of the alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil (Hypera postica) is a well‐known example of a worldwide‐distributed pest with high genetic variation. Based on the mitochondrial genes, the alfalfa weevil clusters into two main mitochondrial lineages. However, there is no clear picture of the global diversity and distribution of these lineages; neither the drivers of its diversification are known. However, it appears likely that historic demographic events including founder effects played a role. In addition, Wolbachia, a widespread intracellular parasite/symbiont, likely played an important role in the evolution of the species. Wolbachia infection so far was only detected in the Western lineage of H. postica with no information on the infecting strain, its frequency, and its consequences on the genetic diversity of the host. We here used a combination of mitochondrial and nuclear sequences of the host and sequence information on Wolbachia to document the distribution of strains and the degree of infection. The Eastern lineage has a higher genetic diversity and is found in the Mediterranean, the Middle East, Eastern Europe, and eastern America, whereas the less diverse Western lineage is found in Central Europe and the western America. Both lineages are infected with the same common strain of Wolbachia belonging to Supergroup B. Based on neutrality tests, selection tests, and the current distribution and diversification of Wolbachia in H. postica, we suggested the Wolbachia infection did not shape genetic diversity of the host. The introduced populations in the United States are generally genetically less diverse, which is in line with founder effects.     

Antennal sensilla of the adult alfalfa weevil,Hypera postica (Gyllenhal) (Coleoptera : Curculionidae)

Sensilla on the antennae of adult alfalfa weevils, Hypera postica (Gyllenhal),were studied with scanning and transmission electron microscopes to determine the sensilla structure and possible chemo- and mechanoreceptive functions for food detection.Five types of sensilla trichodea (s.t.) occur (length 35 – 135 μm); s.t. I (71 – 135-μm long)are thick-walled without radial pores and s.t. II (50 – 75-μm long), the most abundant s.t., are multiporous. Bifurcated and trifurcated types (75 –85 -μm long) also occur. Five types of sensilla basiconica (s.b.) are present and include s.b. I (26 – 50-μm long), a fluted uniporous type with a rugose surface and encircling ledges; s.b. II (15 – 35-gm long), a common multiporous type with many longitudinal grooves; and s.b. IV (4 – 7-μm long), which are 3 apical, short pegs. Sensilla styloconica occur in a small band among the s.b. type II. One or 2 epidermal gland ducts are adjacent to most s.t. and some s.b.     

Digestive amylase and pectinase activity in the larvae of alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive α-amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for α-amylase against starch at acidic pH (pH 5.0). α-Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. α-Amylase activity was significantly decreased in the presence of Ca²⁺, Mg²⁺ and sodium dodecylsulfate. On the contrary, K⁺ and Na⁺ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of α-amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for α-amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.     

Alternatives to key factor analyses for assessing the population dynamics of Hypera postica (Coleoptera: Curculionidae)

Key factors are those causes that are most responsible for the observed changes in population density between generations. Stage-frequency of alfalfa weevil was modeled using Manly-1997 model. Multiple decrement life table parameters from an 8 year-field study were analyzed using traditional and λ contribution methods. In traditional method, the key factor was determined as k _LII, the death of period-two larvae from all mortality factors, whereas in λ contribution method, the key factor was determined as b _2&3, oviposition rate of females per square meter. These differences result directly from the sensitivity of the population growth rate, λ, to variation of both k and b in various stages of the weevil. Among all mortality factors, only k _LI-Z, the death of period-one larvae from the entomopathogenic fungus, Zoophthora phytonomi (Arthur) Batko (Zygomycetes: Entomophthoraceae), k _LII and k _LII-Z, the death of period-two larvae from Z. phytonomi, acted in a density dependent fashion. Remaining factors were independent of population densities affected. Using λ contribution method, life table approach still remains a major way of studying the dynamics of field populations for applied ecologists and population managers.     

An Immunoassay Method for Detecting Microctonus aethiopoides (Hymenoptera: Braconidae: Euphorinae) Parasitism in the Alfalfa Weevil, Hypera postica (Coleoptera: Curculionidae)

A simple immunological assay was developed as an alternative to the dissection/visualization method for detecting the presence of the parasitoid, Microctonus aethiopoides Loan, in the alfalfa weevil, Hypera postica (Gyllenhal). The dot-blot assay was validated using laboratory-reared and field-collected adult weevils of known parasitization status. The dot-blot assay was also used to estimate the developmental stage of the parasitoid within parasitized adult hosts. The assay results can be used to forecast parasitoid emergence dates and estimate the parasitism rate of M. aethiopoides in alfalfa weevil populations.     

Coexistence of two mitochondrial DNA haplotypes in Japanese populations of Hypera postica (Col., Curculionidae)

Abstract:  In Japan, the alfalfa weevil, Hypera postica , was first recorded in 1982 from Fukuoka and Okinawa Prefectures and has been spreading to many other prefectures. The weevil seriously infests the Chinese milk vetch, Astragalus sinicus , one of the most important honey resources for honeybees in Japan. Direct sequencing of partial mitochondrial DNA and PCR-RFLP data for alfalfa weevil individuals indicated the coexistence of two haplotypes at various localities in Japan. Molecular phylogenetic analysis for H. postica haplotypes and strains indicated that the two Japanese haplotypes had not derived from a single genetic origin. Based on the results, special comments are made on biological control measures using introduced parasitic waSPS.     

The attachment and stylostome of Trombidium newelli (Acari: Trombidiidae), an ectoparasitic mite on adults of alfalfa weevil, Hypera postica (Coleoptera: Curculionidae)

Larvae of the mite Trombidium newelli Welbourn and Flessel are ectoparasitic on adult alfalfa weevil, Hypera postica (Gyllenhall), an insect pest of alfalfa. The mite larvae are found under the elytra, attached to the dorsal surface of weevils abdomen. T. newelli larvae use their chelicerae to penetrate the pliable and weakly sclerotized areas of the hosts integument, and to hold on firmly to the host. The attachment sites associated with larval mites of different degrees of engorgement were examined using both light and scanning electron microscopy. The ventral inside surface of parasitized host tergites revealed a characteristic injury as spots varying in color from white-yellow to golden-brown depending on the engorged size of the mite. In addition, spots associated with fully engorged mite larvae showed an aggressive dendritically radiating mass expanding outward from the site of cheliceral penetration. This mass, known as feeding tube or stylostome was branched, with each branch ending in a cluster of closed bulbs. Stylostomes appear to exist independently in the hosts tissue. Stylostome mass corresponded with the engorgement state of the mite, increasing as the mite larva increased in size. The possible nature and mechanism of stylostome formation is discussed.     

Contrasting patterns of molecular diversity and Wolbachia infection in bisexual and parthenogenetic Strophosoma weevils (Coleoptera: Curculionidae)

It has been postulated that parthenogenesis in weevil species is of hybrid origin, but some have speculated that Wolbachia infection plays a role through the modification of host breeding systems. Here we focus on Strophosoma weevils, which are known to be pests in young forest stands. Using molecular data, we investigated the diversity of the two most common Strophosoma species in Europe: S. capitatum, which reproduces bisexually, and S. melanogrammum, which is parthenogenetic. Also researched were their associations with the endosymbiotic bacterium Wolbachia. These species of weevil were found to be clearly distinguishable based on their mitochondrial DNA, with the bisexual taxa being more diverse. However, the nuclear DNA divergence of the two species was very low, and the parthenogenetic taxon was found to be heterozygous. Wolbachia infection was detected in all individuals of the S. melanogrammum populations and less than half of the S. capitatum populations. Moreover, multiple Wolbachia strains were found in both taxa (two in the former and three in the latter). The results of this research suggest that parthenogenesis in this genus is of hybrid origin and that Wolbachia could have played a role in speciation of these weevils.     

New haplotype and inter‐strain reproductive compatibility of Wolbachia‐uninfected alfalfa weevil,Hypera postica (Coleoptera: Curculionidae), in Japan

Hypera postica is a univoltine invasive pest of alfalfa, Medicago sativa, in North America. In Japan, H. postica was first found in 1982 from Fukuoka and Okinawa Prefectures and became a serious pest of Chinese milk vetch, Astragalus sinicus, cultivated as a honey source for humans and green manure for rice. In North America, three strains, Western, Eastern and Egyptian, have been identified and the Western strain is infected with Wolbachia, which causes complete inter‐strain reproductive incompatibility. In contrast, only Western and Egyptian strains had been reported throughout Japan and none of the Western strain examined for the Fukuoka populations in northern Kyushu was infected with Wolbachia. First, we screened populations from northern Kyushu collected since 1982 for geographical and chronological distribution of the Eastern strain. The Eastern strain has been found at low frequencies since 1985 and is still present in 2014. Second, we experimentally tested our hypothesis that inter‐strain crosses between uninfected Western‐strain males and Egyptian‐strain females should produce viable offspring. We crossbred virgin adults reared individually from field‐collected larvae and confirmed that the F₁ eggs of crosses between the Western‐strain males and the Egyptian‐strain females develop successfully into larvae.     

Prevailing triple infection with Wolbachia in Callosobruchus chinensis (Coleoptera: Bruchidae)

Prevailing triple infection with three distinct Wolbachia strains was identified in Japanese populations of the adzuki bean beetle, Callosobruchus chinensis. When a polymerase chain reaction (PCR) assay was conducted using universal primers for ftsZ and wsp, Wolbachia was detected in all the individuals examined, 288 males and 334 females from nine Japanese populations. PCR-restriction fragment length polymorphism (RFLP) analysis of cloned wsp gene fragments from single insects revealed that three types of wsp sequences coexist in the insects. Molecular phylogenetic analysis of the wsp sequences unequivocally demonstrated that C. chinensis harbours three phylogenetically distinct Wolbachia, tentatively designated as wBruCon, wBruOri and wBruAus, respectively. Diagnostic PCR analysis using specific primers demonstrated that, of 175 males and 235 females from nine local populations, infection frequencies with wBruCon, wBruOri and wBruAus were 100%, 96.3% and 97.0%, respectively. As for the infection status of individuals, triple infection (93.7%) dominated over double infection (6.1%) and single infection (0.2%). The amounts of wBruCon, wBruOri and wBruAus in field-collected adult insects were analysed by using a quantitative PCR technique in terms of wsp gene copies per individual insect. Irrespective of original populations, wBruCon and wBruOri (107 -108 wsp copies/insect) were consistently greater in amount than wBruAus (106 -107 wsp copies/insect), suggesting that the population sizes of the three Wolbachia strains are controlled, although the mechanism is unknown. Mating experiments suggested that the three Wolbachia cause cytoplasmic incompatibility at different levels of intensity.     

Identification and biological role of the endosymbionts Wolbachia in rice water weevil (Coleoptera: Curculionidae)

Wolbachia spp. are obligate intracellular bacteria present in reproductive tissues of many arthropod species. Wolbachia infection status and roles in host reproduction were studied in the rice water weevil, Lissorhoptrus oryzophilus Kuschel (Coleoptera, Curculionidae), an introduced species in China. We examined Wolbachia infection status in five populations in China where it reproduces parthenogenetically, and one native population in Southeast Texas, where it reproduces bisexually. All populations were infected by Wolbachia, and all specimens in each population were infected by Wolbachia of a single strain. Phylogenetic analyses based on multilocus sequence typing system indicated that Wolbachia in non-native L. oryzophilus weevils diverges evidently from those in native weevils. After treatments with tetracycline, parthenogenetic weevils reduced the fecundity significantly and eggs were not viable. Our results suggest that Wolbachia are necessary for oocyte production in L oryzophilus.     

Wolbachia infection in the Korean endemic firefly,Luciola unmunsana (Coleoptera: Lampyridae)

Wolbachia is one of the most prevalent endosymbiontic bacteria of arthropods. The bacterium induces sex ratio distortions in various host insects through processes such as cytoplasmic incompatibility, feminization, male killing, and parthenogenesis. We investigated if the Korean endemic firefly, Luciola unmunsana was infected with the bacterium because the species had an abnormal sex ratio in the field. The results show that some individuals are infected with the bacterium. Phylogenetic analyses showed that the bacterial strain infecting the firefly is closely related to strains that infect phylogenetically distant hosts.     

Sensilla on the antennae,mouthparts, and body of the larva of the alfalfa weevil,hypera postica (gyllenhal) (Coleoptera : Curculionidae)

The morphology of the antennal complex, major mouthpart sensilla and body sensilla of alfalfa weevil larvae, Hypera postica (Gyllenhal) (Coleoptera : Curculionidae), is described. All instars have a short 1-segmented antenna with a large, multiporous sensillum basiconicum (s.b.) on its apex. Surrounding the s.b. are 2 unusual sensilla auricularia, and 3 small s.b., 2 of which are uniporous. The galea of the maxilla contains 11 s.b. and the apex of the palpus bears 12 thick-walled, uniporous s.b. The labial palpus has 10 similar s.b. apically.First-instar larvae have long, rodlike sensilla with a large, thick-walled bulbous apex; they are probably mechanoreceptors. The other 3 instars bear unusual nodulated and ridged sensilla with thin walls and a fragment-filled lumen. Sensilla on the apex of the thoracic legs and prolegs are also illustrated.     

Taxonomic revision of Melanocyphus Jekel (Coleoptera: Curculionidae)

Cornops aquaticum (Bruner) is host specific on Eichhornia spp. and Pontederia spp. (Pontederiacae). Its present distribution ranges from Mexico to Argentina. The number of juvenile instars (5-7) apparently mirrors the photoperiod and temperature pulses of different climatical conditions in the respective geographical regions. Based on life-history data, three working hypotheses are proposed for forthcoming studies. These will test whether the varying number of juvenile instars represents a phenotypic plasticity of a single genotype or an adaptation that is genetically fixed, due to an evolutionary relationship of the host with its host-plant.     

Responses of Chalcidoidea (Hymenoptera) parasitoids to invasion of the cabbage seedpod weevil (Coleoptera: Curculionidae) in western Canada

Invasion of the European weevil, Ceutorhynchus obstrictus (Marsham), was investigated through surveys of its range and population densities in Alberta and Saskatchewan from 2001 to 2005. After it was first reported in southern Alberta, C. obstrictus rapidly expanded its range and abundance. Our more recent surveys indicate that its northward expansion has slowed, but that it has continued to extend its range eastward to southcentral Saskatchewan. The distribution and abundance of parasitoids of C. obstrictus in Alberta and Saskatchewan were investigated from 2003 to 2005 by mass rearing canola pods infested with C. obstrictus larvae. Although weevil populations were not parasitized for several years immediately following its introduction to southern Alberta, a surprisingly diverse assemblage of Chalcidoidea parasitoids, comprising 12 species from four families, were recently reared from weevil-infested canola siliques in Alberta and Saskatchewan. The Chalcidoidea fauna of C. obstrictus include species with both Nearctic and Holarctic distributions, with some species having restricted host ranges and others that are more niche than taxon-specific. These Chalcidoidea species appear to have expanded their host ranges to parasitize C. obstrictus in the region. Most parasitism is attributable to Trichomalus lucidus (Walker), Chlorocytus sp., and Pteromalus sp. (Pteromalidae), and Necremnus tidius (Walker) (Eulophidae). Parasitism levels varied considerably over the three years of this study. From 2003 to 2005 increases in parasitism occurred among all four of the species dominating the parasitoid fauna of C. obstrictus, but greater increases were observed for Chlorocytus sp. and Pteromalus sp. than for T. lucidus. Parasitoid species have sometimes caused substantial levels of host mortality, although current levels are usually less than 15% for all species combined and so are not sufficient to control weevil populations. Implementing a classical biological control program for C. obstrictus by reconstructing its European natural enemy complex is being considered, but it is still uncertain whether parasitism levels by native Chalcidoidea will increase over time since considerable year-to-year variation has been found. Parasitism levels of C. obstrictus should therefore continue to be monitored to assess whether a classical biological control program should be implemented.     

Mating behavior ofBathyplectes curculionis [Hym.: Ichneumonidae] a parasitoid of the alfalfa weevil,Hypera postica Col.: Curculionidae]

We describe the courtship behavior of male and femaleBathyplectes curculionis (Thomson). FemaleB. curculionis are polygamous and sexually receptive for up to 2 days after eclosion. The high activity levels of virgin females combined with their polygamy and long period of sexual receptivity allow for post-dispersal mating which may facilitate maximum gene flow between localB. curculionis populations. This high genetic diversity may be partially responsible for the widespread occurrence ofB. curculionis in North America.

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Résumé Nous décrivons le comportement sexuel deBathyplectes curculionis (Thomson) male et femelle. Les femelles deB. curculionis sont polygames et réceptives 2 jours après l'éclosion. L'activité des femelles vierges et leur polygamie associées à la longue période de réceptivité sexuelle permettent un maximum d'échanges génétiques entre les populations locales. Cette grande diversité génétique peut être en partie responsable de l'étendue de la distribution deB. curculionis en Amérique du Nord.

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Partially supported by funds from both the Department of Entomology and the Graduate School of The Ohio State University.

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Submitted to the Graduate School of The Ohio State University in partial fulfillment of the requirements of the degree of Doctor of Philosophy. Present address: Agricultural Research Center, University of Florida, 3205 S. W. 70th Avenue, Ft. Lauderdale, Florida, U.S.A., 33314.     

New Weevils (Coleoptera: Curculionidae) from Rovno Amber

Paleontological Journal - A new genus Rovnoslonik Legalov, Nazarenko and Perkovsky gen. nov. (type species Rovnoslonik damzeni Legalov, Nazarenko and Perkovsky sp. nov.; tribe Stromboscerini,...