Leslie L. Allee

Genetic differences in allozymes and in formation of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae

Abstract In this study, we examined variation among isolates of the entomopathogenic fungus Metarhizium anisopliae in their ability to germinate and produce infection structures (appressoria) in different nutrient media. We also examined allozyme variation within and among 120 isolates of Metarhizium spp. Electromorph differences were noted and genotypic designations were assigned for eight loci encoding the following enzymes: glucose phosphate isomerase, glutamate dehydrogenase, glutamic pyruvic transaminase, glutathione reductase, malate dehydrogenase, phosphogluconate dehydrogenase, peptidase with glycyl leucine, and triosephosphate isomerase. Forty-eight distinct genotypic classes were found. Only the thirteen M. anisopliae var. majus isolates and an isolate of Metarhizium flavoviride possessed multibanded phenotypes characteristic of heterozygous genotypes. Three isolates were heterozygous at one locus, seven isolates at two loci, and four isolates at four loci. Based on the level of genetic similarities between the three recognized species ( M. anisopliae, Metarhizium album , and M. flavoviriae ) we report that isolates currently assigned as M. anisopliae are a strongly heterogenous array of multilocus genotypes that includes five varieties and two undescribed cryptic species. Genetic similarities provided evidence for geographical clustering. For example, Brazil and Colombia contain a common, widely dispersed genotypic class; eight additional classes are more locally distributed. Nutrient requirements for germination and appessorium formation were frequently host related, irrespective of genetic and geographical distance. Most of those lines of M. anisopliae var. anisopliae and var. majus isolated from scarabaeids germinated readily only in the presence of a crude protein/chitin product. Lines isolated from other coleopterans were much more adaptable and produced appressoria in yeast extract media. Unlike the majority of isolates from other hosts, lines isolated from Hemiptera usually germinated well in media containing glucose as the sole carbon source, and glucose allowed formation of appressoria. The role of selectable strain variations in the development of pathogen strategies is discussed.

World-wide distribution of genetic variation among isolates of Beauveria spp.

Genetic variation among isolates of Beauveria spp. was defined by an analysis of electrophoretically demonstrable allelic variations of allozyme coding genes. One hundred and forty-six isolates from diverse geographical locations were assigned to forty-seven distinctive genotypic classes. Clustering analysis demonstrated that four morphological species ( B. brongniartii, B. vermiconia, B. caledonica and Tolypocladium cylindrosporum ) were genetically distinct from each other. However, with the exception of B. brongniartii , which clustered separately with B. amorpha , they were not clearly resolved from lines of B. bassiana . The level of genetic distance shown between cluster groups of B. bassiana indicated that it represents a species aggregate, components of which display overlapping genetic variability with isolates currently assigned to other species. All isolates were homozygous at each locus, consistent with haploidy, except for B. caledonica , which was heterozygous at one of the four loci. In spite of the maintenance of high diversity in B. bassiana , three geographically widespread genotypic classes were found to contain the majority of isolates. This suggests that, in many situations, B. bassiana exists with a clonal population structure. Various other aspects of the allozyme data (the magnitude of genetic distances between populations, gene diversity, and the pattern of distribution of genotypic classes) indicate that chromosomal recombination between different genotypes of B. bassiana spp. is rare or absent.

Germination and infection processes of the entomophthoralean fungus Erynia radicans on the potato leafhopper, Empoasca fabae

The germination and penetration processes of Erynia radicans on fifth instar Empoasca fabae nymphs at 20°C, 100% relative humidity, were investigated by scanning electron and fluorescence microscopy. Oval primary and secondary conidia attached to the host cuticle and germinated rapidly (60% within 2 hr). While percentage germination did not differ on the three body regions of the host, significant differences were observed in the modes of germination. The percentage of spores that produced germ tubes increased from 10% on the leafhopper head to 13% on the thorax and 22% on the abdomen, while capilliconidiophore production decreased from 88 to 84 to 71% on the respective body regions. Hyphae from spores deposited on sclerites displayed strong directional growth toward interscleral membranes, especially on the host abdomen. Penetration of the cuticle was effected by formation of appressoria. Small, globular appressoria were formed immediately adjacent to spores or on short, nonseptate germ tubes, while large, elongate appressoria were produced usually on long, but occasionally on short, septate hyphae. Approximately 3% of penetrations occurred on the leafhopper head, 16% on the thorax, and 74% on the abdomen; ca. 61% of all penetrations occurred through membranes, especially in the intersegmental folds of the abdomen. Earliest penetrations were observed 3–4 hr after inoculation, but significant numbers of penetrations were not observed until after 6 hr. The number of penetrations on individual leafhoppers was largely independent of dose (mean r2 = 0.174), indicating substantial variability in susceptibility of individual leafhoppers. The percentage of viable oval conidia which ultimately gave rise to penetrations (either directly or indirectly via secondary spore production) was ca. 5% after 10–12 hr and 20% after 48 hr. The percentage of leafhoppers infected (percentage penetrated by one or more hyphae from a mean dose of 41 spores per insect) increased rapidly from only 2.4% at 4 hr postinoculation to ca. 60% after 8 hr. The LD50 was estimated at 4.1 spores per leafhopper.

Infection by Beauveria bassiana of Leptinotarsa decemlineata larvae as a consequence of fecal contamination of the integument following per os inoculation.

A study ofper os inoculated larvae was undertaken to determine if the entomopathogenic fungus,Beauveria bassiana, is able to infect the Colorado potato beetle,Leptinotarsa decemlineata, via the alimentary tract. Surface orper os inoculated larvae which were immediately surface sterilized post inoculation did not succumb to infection, whereas those larvae not sterilized became infected. Histological studies of fed or starved, agnotobiotic (with microbial flora) and axenic larvae revealed that conidia can germinate in the gut regardless of the presence of gut microflora. However, infection via the alimentary tract was never observed in fed larvae and only noted in a single starved individual. It is concluded that infections ofper os inoclated larvae occurred after surface contamination of the integument by viable conidia contained in the frass. The rate of food passage through the gut is probably important in preventingper os infections.

Lady Beetles in New York: Insidious Invasions, Erstwhile Extirpations, and Recent Rediscoveries

Abstract Over the past 40 years, the complex of coccinellid (lady beetle or ladybug) species in New York has undergone substantial changes. Primarily, these changes have involved the decline of native species and the increase and spread of adventive species. Species declines have proceeded to the extent that several native species were feared to be extirpated from New York. Here we report that two of these native species, Adalia bipunctata (Two-spotted Lady Beetle) and Coccinella novemnotata (Nine-spotted Lady Beetle), were rediscovered in New York, in 2009 and 2011, respectively, by volunteers and specialists working for the Lost Ladybug Project. We found that the current coccinellid complex in New York is significantly less diverse and has a significantly higher proportion of foreign species compared to the complex in the past. We discuss the potential causes and implications for these shifts and rediscoveries.

The Status of Coccinella undecimpunctata (L.) (Coleoptera: Coccinellidae) in North America: An Updated Distribution from Citizen Science Data

Coccinella undecimpunctata L. is an Old World aphid predator that is native to central Asia, North Africa, Iceland, and much of Europe. The species was introduced accidently (Gordon 1985) and first reported in North America by Schaeffer (1912) based on two specimens from near Boston, Massachusetts. Since then, the species was found again in Massachusetts (Dobzhansky 1931), Vermont (Parker et al. 1976), Maine and New Jersey (Belicek 1976), and Ohio (Dailey et al. 1978) in the eastern USA and in Canada in New Brunswick, Prince Edward Island, and Quebec (Brown 1940), Nova Scotia and Newfoundland (Chapin 1956), and Ontario (Brown 1962). By the 1960s, C. undecimpunctata had also been found in the Pacific Northwest, near Seattle, Washington (Russell 1968), Vancouver, British Columbia (Belicek 1976) and Corvallis, Oregon (Wheeler and Hoebeke 1981). Wheeler and Hoebeke (1981) also added Connecticut, New York, Pennsylvania, and Rhode Island to update the distribution of C. undecimpunctata and noted the importance of this predator’s role in pest suppression. On Long Island, C. undecimpunctata had been found to represent 55% of all coccinellids found on potatoes (Day 1965). It has been proposed that C. undecimpunctata successfully extended its range with the help of the St. Lawrence Seaway as well as the railroad (Larochelle 1979; Watson 1979; Wheeler and Hoebeke 1981). But following a period of increase in the mid-20th century, the species appears to have declined to the point of extirpation from much of its former North American range. Wheeler and Hoebeke (2008) posed the question of whether or not C. undecimpunctata was still present in North America. Those authors reported that no additional USA states or Canadian provinces had been added to the distribution of C. undecimpunctata since the 1980s and documented the lack of recent records of C. undecimpunctata in Ontario, Québec, the Atlantic Provinces of Canada, New England, the midAtlantic states of Pennsylvania, Delaware, Maryland, and Virginia, and New York State. They suggested that C. undecimpunctata may persist in low numbers in some parts of the Northwest and Northeast of the USA, and in southwestern British Columbia and the eastern Maritime Provinces of Canada; they invited anyone who encountered this species to document the finding. Here we confirm the presence ofC. undecimpunctata in the Pacific Northwest and lend further evidence to its possible extirpation elsewhere in North America. Two C. undecimpunctata were located through postings to BugGuide.net – a continuing online collection of insect photographs curated by a community of naturalists. Twelve more C. undecimpunctata were located through submissions to the Lost Ladybug Project (www. lostladybug.org) a continent-wide coccinellid survey funded by the National Science Foundation since 2008. The Lost Ladybug Project is a webbased, citizen science program in which participants provide a digital photograph of the insect, contact information, location details, and habitat information via an online form. All specimens are photo-vouchered and identifications are made by researchers at Cornell University. During the five years between 2008 and 2012, the Lost Ladybug Project has received and identified over 20,000 photographic specimens. This relatively inexpensive method of data collection has permitted an abundance of data to be collected over a broad geographic range. Much as predicted by Wheeler and Hoebeke (2008), all 14 recently located C. undecimpunctata were found in the Pacific Northwest: Coquitlam, British Columbia; Puyallup and Bothell, Washington; Salem and Scio, Oregon. The Lost Ladybug Project has not received any reports from two eastern locations suggested by Wheeler and Hoebeke (2008) where C. undecimpunctata may persist: Sable Island and Brier Island, both in the Maritime Provinces of Canada.

Nuclear events during germination and appressorial formation of the entomopathogenic fungus Zoophthora radicans (Zygomycetes: Entomophthorales)

Abstract Conidial germination, appressorial formation, and secondary sporulation in Zoophthora radicans occurred independently of DNA replication and nuclear division. Video-image analysis combined with epifluorescence microcopy showed that at 18 hr postinoculation, approximately 10% of germlings had undergone the first DNA duplication and nuclear division and that 20% of germlings had formed appressoria. Hydroxyurea, an inhibitor of DNA synthesis, prevented DNA replication and nuclear division but allowed production of germ tubes and appressoria. Actinomycin D, an inhibitor of RNA synthesis, totally inhibited germination at concentrations above 10 μg/ml. At lower concentrations, actinomycin D allowed formation of germ tube and appressoria in a dose-dependent fashion. However, even at the lowest concentration tested (1 μg/ml), nuclear division and formation of penetration hyphae were prevented. Formation of secondary conidia and capilliconidia was also independent of nuclear division but was totally inhibited by drugs blocking RNA or protein synthesis. The implications of differentiation without prior nuclear division are discussed.

NEW RECORDS OF COCCINELLIDAE (COLEOPTERA) FROM WYOMING, USA

Faunal distributions are dynamic, and the fauna associated with a particular area may change due to accidental or intentional introductions of new species, extinctions, and natural geographic range expansions or contractions of species (Brodman et al. 2002; McCorquodale and BondrupNielsen 2004). Thus, periodic field surveys and collections of fauna are necessary to maintain a current species inventory. Accordingly, ongoing curation of new material deposited into the zoological collections of museums and the periodic review of these collections is needed for generating accurate, up-to-date faunal lists (McCorquodale and Bondrup-Nielsen 2004; Hesler and Kieckhefer 2008). A comprehensive overview of the lady beetle (Coleoptera: Coccinellidae) fauna of North America north of Mexico was provided by Gordon (1985). Several updates to the fauna have been published, including those for particular states within the USA and provinces of Canada. For instance, Michels (1987) provided a checklist of coccinellids and reported 16 new species records of coccinellids from Wyoming, USA. Hesler and Kieckhefer (2008) suggested that examination of undetermined material in beetle collections may yield additional species records of coccinellids. Recently, undetermined beetles within the University of Wyoming Insect Museum, Laramie, Wyoming were examined. Keys in Gordon (1985) and known geographic distributions presented in Gordon (1985) and Michels (1987) were used to determine new records for four species of coccinellids from Wyoming among the undetermined material. This paper summarizes those new records. A revised classification of the Coccinellidae (Seago et al. 2011) is used.