Size-specific susceptibility of the pest slugs Deroceras reticulatum and Arion lusitanicus to the nematode biocontrol agent Phasmarhabditis hermaphrodita

The nematode Phasmarhabditis hermaphrodita is a commercially available biocontrol agent against slugs. This product is especially interesting for use in organic farming, where products containing metaldehyde or carbamates cannot be used for controlling pest slugs. We investigated the potential of P. hermaphrodita for the control of the pest slugs Deroceras reticulatum and Arion lusitanicus. These two species are the most harmful slug pests in Switzerland. At different times of the year, we collected slug specimens of different weight and assessed their susceptibility to P. hermaphrodita in the laboratory. Batches of five slugs were subjected to five different doses of nematodes plus an untreated control and replicated three times. During six weeks, feeding and survival of the slugs were recorded. D. reticulatum was strongly affected by increasing nematode doses, irrespective of the slugs' body weight. In small specimens of A. lusitanicus, feeding and survival were strongly affected by the nematodes, while larger specimens remained almost unaffected. Because A. lusitanicus has an asynchronous development in Switzerland, it seems difficult to control the entire population with a single nematode application. To what extent nematodes will be used in practice for slug control depends on their effectivity against the pest slugs of major importance, on the longevity of the molluscicidal effect and on the price of nematodes.     

Cold tolerance and freeze-induced glucose accumulation in three terrestrial slugs

Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about − 4 °C when cooled under dry conditions, but freezing of body fluids was readily induced at − 1 °C when in contact with external ice crystals. All three species survived freezing for 2 days at − 1 °C, and some A. rufus and A. lusitanicus also survived freezing at − 2 °C. ¹H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22 μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates.     

Relationships between temperature, development and survival of different life stages of the tomato fruit fly, Neoceratitis cyanescens

The development and survival of female Neoceratitis cyanescens (Bezzi) (Diptera: Tephritidae) from egg to complete ovarian maturation were studied in the laboratory at five different constant temperatures: 15, 20, 25, 30, and 35 °C. The aim of this study was to get information on the influence of temperature on pre-mature stages, as a prerequisite to optimise rearing procedures and to understand temporal and geographical patterns of fruit fly occurrence. The developmental rate of the different life stages increased linearly with increasing temperatures up to 30 °C. The fastest development of pre-mature stages was recorded at 30 °C (22±1 days) and the slowest at 15 °C (98±3 days). The day-degrees requirements (K) to complete total development were 432.6 day-degrees. Lower temperature thresholds were 11.4, 11.9, 10.0, and 11.1 °C for egg, larval, pupal stages and ovarian maturation, respectively. The number of adults obtained from an initial batch of 100 eggs reached a maximum (64) at 25 °C. At 35 °C, no adults emerged. Larval developmental time was significantly shorter in green tomato fruits than in potato tubers at 15, 20, and 25 °C. Mortality rate of larvae was higher in green tomato fruits than in potato tubers at 25 and 30 °C.     

Phylogeny of slug species of the genus Arion: evidence of monophyly of Iberian endemics and of the existence of relict species in Pyrenean refuges

The Iberian Peninsula contains the majority of the Paleartic land slug species of the genus Arion, which exhibits diverse taxonomic problems. The present study investigated Arion taxonomy on the basis of analyses of the mitochondrial ND1 gene and nuclear internal transcribed spacer 1 (ITS1) sequences. The Iberian endemic species were monophyletically clustered in two divergent sister clades. The topotype specimens of Arion lusitanicus and the closely related species Arion nobrei and Arion fuligineus, as well as Arion hispanicus and Arion flagellus, were grouped into an ‘Atlantic’ clade, whereas Arion baeticus, Arion gilvus, Arion anguloi, Arion wiktori and Arion paularensis were included in a ‘Continental–Mediterranean’ clade. Calibration of mutation rate in the ND1 gene suggested that the divergence of these two clades occurred around the Pliocene–Pleistocene boundary, with subsequent speciation events during the Pleistocene. A group of ancestral and divergent endemic species with distribution centred in the Pyrenean mountain range (Arion molinae, Arion lizarrusti, Arion antrhacius and Arion iratii) arose in the Pliocene and survived through the Pleistocene in geographically confined small populations. Arion lusitanicus showed up to be polyphyletic: specimens, sampled outside the geographic range of the topotype in the north‐western Iberian Peninsula, were included in a non‐monophyletic clade together with the widely distributed species Arion ater and Arion rufus. The divergent species with a wide European distribution (Arion subfuscus, Arion hortensis, Arion fagophilus and Arion intermedius) were located in basal positions in all topologies. The evolutionary history of these slug species (highly sensitive to climatic factors, with capacity for both outcrossing and selfing, and with low dispersal ability) appears to have been moulded by Pliocene–Pleistocene climate events and by the rugged topography of southern Europe, giving rise to repeated cycles of population isolation during periods of glaciation alternating with interglacial expansions limited by geographic barriers.     

Effect of freeze temperature on ice formation and long-term survival of the woolly bear caterpillar (Pyrrharctia isabella)

Tissue ice content and post-freeze survival were documented for caterpillars of the arctiid moth Pyrrharctia isabella. Tissue ice content was inversely dependent on freeze temperature (−3 °C=24.4%, −6 °C=40.2%, −10 °C=48.7%) but values were substantially less than expected given hemolymph osmolality. Accumulation of glycerol (200-300 mM) in the hemolymph helped to colligatively reduce the amount of freezable water. Caterpillars engaged in locomotion within minutes after thawing but mortality occurred over the ensuing weeks, with the highest level (52.2%) occurring in the −10 °C fast thaw group. Pupation rates ranged between 45.7 to 52.4% of caterpillars in a test group. Adult emergence exceeded 60% of the pupae in the -3 and -6 °C test groups. Hence, P. isabella caterpillars survived ecologically relevant freezes and continued their life cycles to adulthood.     

The effects of the slug biological control agent, Phasmarhabditis hermaphrodita (Nematoda), on non-target aquatic molluscs

The nematode Phasmarhabditis hermaphrodita is used as a commercial biological control agent of slugs in the UK. Although it is known to affect other terrestrial mollusc species, its effects on freshwater molluscs are not known. The present study investigated the effects of P. hermaphrodita on the survival of juvenile Lymnaea stagnalis and Physa fontinalis, two common freshwater snails, at 'spray tank' concentration and a 50% diluted 'spray tank' concentration over a 14-day period. Survival of L. stagnalis was significantly reduced at both application levels but P. fontinalis suffered no mortalities over the experimental period. The possible differential mechanisms of pathology between the two host species are discussed.     

Resource allocation in a simultaneously hermaphroditic slug with phally polymorphism

The theory of resource allocation assumes that a resource not allocated to one function may be reallocated to another. Thus, in hermaphroditic species, an individual that suppresses the use of one sex function may free resources for the other sex function. We determined the relative importance of male copulatory organs in terms of their fraction of the total dry body weight and tested whether in the pulmonate land slug Deroceras laeve (Müller), individuals that lack the male copulatory organs (aphallics) reallocate this resource towards the female structures and/or towards life-history traits. To this end, we raised 13 families under uniparental reproduction and compared growth, length of the juvenile period, number of eggs produced, percentage of hatched eggs and hatching time among a- and euphallics. We also measured the reproductive and sex allocation of all individuals. Six out of 13 families contained no euphallic individuals. In the other seven families, the proportion of euphallic individuals ranged from 0.13 to 0.43. There was an enormous variation in life-history traits and reproductive and sex allocation among individuals, even among individuals of the same family. Allocation to the male function was very low in euphallic slugs (i.e. 1.35% of the total body dry mass and 12.33% of the total reproductive dry mass). Our results did not reveal a reallocation from the lost male function towards the female function, nor towards one of the life-history traits. Finally, we propose a scenario that could explain the maintenance of phally polymorphism in D. laeve.     

Floral rewards in someScrophularia species (Scrophulariaceae) from the Iberian Peninsula and the Balearic Islands

The genusScrophularia in the Iberian Peninsula and Balearic Islands comprises two sections,Scrophularia andCaninaG. Don. Analyses were carried out on flower production, flower duration and their sexual phases, pollen and nectar production together with observations on their pollinators. Nectar production is correlated with corolla size and pollen production with anther size. The taxa of sect.Scrophularia show greater nectar and pollen production than those of sect.Canina. Also, those of the first section produce more ovules per ovary than those of the second group, production being correlated with the ovary size.Some observations on floral and reproductive biology in some species ofScrophularia from the Iberian Peninsula and the Balearic Islands. I.     

Population Genetics and Identity of an Introduced Terrestrial Slug: Arion subfuscus s.l. in the North-east USA (Gastropoda, Pulmonata, Arionidae)

Several European species of the terrestrial slug genus Arion have been introduced into North America. A case in point is the species complex A. subfuscus s.l. which has become one of the most abundant slug taxa in North America. In Europe this complex consists of at least two cryptic species, viz. A. fuscus and A. subfuscus s.s., the latter of which is further subdivided in five strongly divergent mtDNA lineages (A. subfuscus S1–S5). In order to determine which of these A. subfsucus s.l. taxa are present in the NE USA and in order to assess their population genetic structure, we compared mtDNA, nDNA and allozyme variation between populations from the NE USA and Europe. Our results show that (1) at least A. subfuscus S1 has become successfully established in the NE USA, (2) founder effects are the most likely explanation for the loss of a large amount of molecular genetic variation in populations from the NE USA (i.e. a loss of 96% of the 16S rDNA haplotypes, 67% of the ITS1 alleles and 46% of the alleles at polymorphic allozyme loci), and (3) part of the remaining genetic variation in NE USA populations was probably due to multiple introductions from the British Isles and the European mainland, and the hybrid structure of most of these source populations. Apparently, the extreme loss of molecular genetic variation in this introduced species has not prevented it from successfully establishing and spreading in novel environments.     

Reproductive biology of twoPrunus species (Rosaceae) in the Northwest Iberian Peninsula

We studied the reproductive biology ofPrunus spinosa andPrunus mahaleb (Prunoideae, Rosaceae) in the northwest Iberian Peninsula. The two species flowered at the same time (peaking on March 9 and 11, respectively in 1990) but differ significantly in their fruit maturation times. Nectar volume peaked in the early morning in both species, and was ten times greater inP. spinosa than inP. mahaleb. Neither species shows apomixis, nor does fruit-set occur if pollinators are excluded. In both species self-pollination resulted in fewer fruits than open pollination. The principal pollinators belong to theApidae family (79% and 63% of visits toP. spinosa andP. mahaleb, respectively). Results are compared with those for other rosaceous plants with fleshy fruits.     

Effect of temperature on the duration of larval and pupal stages of two species of sciomyzid flies, predators of the snail Lymnaea truncatula

The duration of larval and pupal stages of two species of predatory flies was investigated at five constant temperatures. Ilione albiseta (Scopoli): Mean duration of first and second instars was shortest at 23°C and there was 100% survival of first instar larvae at all temperatures. Mean third instar larval duration decreased from 104 days at 17°C to 27.5 days at 26°C. Total percentage larval survival was greatest at 17°C and least at 26°C. Mean pupal duration decreased as temperature increased and this occurred also under outdoor conditions.Pherbellia cinerella (Fallén): As temperature increased mean larval duration decreased but the percentage of larvae pupating also decreased (100% at 14°C, 33.3% at 26°C). There was a trend for shorter mean pupal durations at higher temperatures.

---

Résumé L'action de la température a été examinée à 14, 17, 20, 23 et 26°C. Les 2 espèces sont prédatrices de L. truncatula, vecteur de la douve du foie en Irlande, où elle provoque à l'àgriculture une perte évaluée à 10 millions de livres par an. Les données recuellies en conditions contrôlées du laboratoire doivent permettre de définir les conditions optimales pour l'élevage continu de ces espèces. Ceci est indispensable pour évaluer, dans la nature leurs potentialités pour la lutte biologique.Ilione albiseta: C'est à 23°C que le développement des larves de premier et second stades sont les plus brefs; la survie du premier stade est totale à toutes les températures constantes examinées. La durée moyenne du 3ème stade décroît de 104 jours à 17°C jusqu'à 27,5 jours à 26°C. Le taux de survie total est plus élevé à 17°C qu'à 26°C. La durée moyenne de la nymphose diminue quand la température croît, et ceci se produit aussi à la température ambiante extérieure. On réduit de moitié la durée du développement de l'éclosion de l'oeuf à l'apparition de l'adulte, entre la nature et le laboratoire, en utilisant respectivement: 23°, 17° et 26°C pour les premier et second stades, le troisième stade, la nymphé. Cela devrait aider à multiplier la souche pour la lutte biologique.Pherbellia cinerella: Avec élévation de la température la durée du développement larvaire diminue, mais aussi le nombre de larves parvenant à la pupaison (100% à 14° et 33,3% à 26°C). La nymphose tendait à être plus brève aux températures les plus élevées.

     

Settlement, early growth and survival of Haliotis rubra in response to different algal species

Five diatom species were isolated from settlementplates at Southern Ocean Mariculture, Victoria,Australia (Navicula sp., Naviculajeffreyi, Cylindrotheca closterium, Cocconeis sp., Amphora sp.) and tested insettlement experiments with Haliotis rubralarvae. Settlement was very low on single speciesdiatom films and varied between 1%–6%. Depending onthe species combination larvae preferred to settle onfilms with mixed diatom species than single speciesfilms. The highest settlement was achieved with amixed film of Navicula sp. and Amphora sp.Five and ten-day-old germlings of Sporolithondurum induced settlement of the abalone Haliotisrubra. However, the settlement rate was significantlylower on germlings than on the whole thallus of thealga. Germlings inoculated with the diatom Navicula sp. induced higher settlement than films ofthe diatom species alone. High settlement of up to52% was also achieved with germlings of the greenalga Ulvella lens. Settlement was reduced onU. lens squares inoculated with the diatom Navicula sp. but higher than on films of the diatomalone. The settlement rate was higher if plates withU. lens were previously grazed by juvenileabalone.Post-larval growth rates were higher on monospecificdiatom films than on U. lens or on S.durum. The best growth rate was obtained with Navicula sp. U. lens and S. durum areboth good settlement inducers, but are notsufficient to support rapid growth of young H.rubra post-larvae. Survival was low on U. lensand on the diatom C. closterium. We suggest thatcommercial nursery plates seeded with U. lenswill result in high and consistent settlement, whilean inoculum with Navicula sp. will ensuresufficient food for rapid growth of the post-larvae.     

Oxidative stress and antioxidant capacity of a terrestrially hibernating hatchling turtle

Hatchlings of the painted turtle, Chrysemys picta, hibernate terrestrially and can survive subfreezing temperatures by supercooling or by tolerating the freezing of their tissues. Whether supercooled or frozen, an ischemic hypoxia develops because tissue perfusion is limited by low temperature and/or freezing. Oxidative stress can occur if hatchlings lack sufficient antioxidant defenses to minimize or prevent damage by reactive oxygen species. We examined the antioxidant capacity and indices of oxidative damage in hatchling C. picta following survivable, 48 h bouts of supercooling (−6°C), freezing (−2.5°C), or hypoxia (4°C). Samples of plasma, brain, and liver were collected after a 24 h period of recovery (4°C) and assayed for Trolox-equivalent antioxidant capacity (TEAC), thiobarbituric acid reactive substances (TBARS), and carbonyl proteins. Antioxidant capacity did not vary among treatments in any of the tissues studied. We found a significant increase in TBARS in plasma, but not in the brain or liver, of frozen/thawed hatchlings as compared to untreated controls. No changes were found in the concentration of TBARS or carbonyl proteins in supercooled or hypoxia-exposed hatchlings. Our results suggest that hatchling C. picta have a well-developed antioxidant defense system that minimizes oxidative damage during hibernation.     

Effects of dehydration rate on physiological responses and survival after rehydration in larvae of the anhydrobiotic chironomid

Strategies to combat desiccation are critical for organisms living in arid and semi-arid areas. Larvae of the Australian chironomid Paraborniella tonnoiri resist desiccation by reducing water loss. In contrast, larvae of the African species Polypedilum vanderplanki can withstand almost complete dehydration, referred to as anhydrobiosis. For successful anhydrobiosis, the dehydration rate of P. vanderplanki larvae has to be controlled. Here, we desiccated larvae by exposing them to different drying regimes, each progressing from high to low relative humidity, and examined survival after rehydration. In larvae of P. vanderplanki, reactions following desiccation can be categorized as follows: (I) no recovery at all (direct death), (II) dying by unrepairable damages after rehydration (delayed death), and (III) full recovery (successful anhydrobiosis). Initial conditions of desiccation severely affected survival following rehydration, i.e. P. vanderplanki preferred 100% relative humidity where body water content decreased slightly. In subsequent conditions, unfavorable dehydration rate, such as more than 0.7 mg water lost per day, resulted in markedly decreased survival rate of rehydrated larvae. Slow dehydration may be required for the synthesis and distribution of essential molecules for anhydrobiosis. Larvae desiccated at or above maximum tolerable rates sometimes showed temporary recovery but died soon after.     

Susceptibility and immune response of Deroceras reticulatum, Milax gagates and Limax pseudoflavus exposed to the slug parasitic nematode Phasmarhabditis hermaphrodita

We exposed three slug species (Deroceras reticulatum (Müller), Milax gagates (Draparnaud) and Limax pseudoflavus L.) to the parasitic nematode Phasmarhabditis hermaphrodita Schneider. P. hermaphrodita was able to cause mortality and feeding inhibition to both D. reticulatum and M. gagates but did not negatively affect L. pseudoflavus. On dissection of surviving L. pseudoflavus large numbers of P. hermaphrodita were found encapsulated in the shell of the slug. We found that by increasing shell size, the slug was able to trap invading nematodes, which could be an immune response to P. hermaphrodita invasion. This is the first report of a slug defense mechanism to inhibit P. hermaphrodita.