Lysosomal neutral red retention time as a biomarker of organophosphate exposure in the earthworm Aporrectodea caliginosa: laboratory and semi-field experiments

Aporrectodea caliginosa is the most common endogeic (topsoil) earthworm in New Zealand and, because of its habitat, is potentially vulnerable to surface-applied pesticides. Lysosomal damage to earthworms, which can be visualized by the use of the neutral red retention assay (NRRA) has been evaluated in this species as a biomarker of organophosphate exposure. Earthworms were exposed in the laboratory to sub-lethal concentrations of chlorpyrifos and diazinon. In a semi-field experiment, earthworms were placed in mesocosms in a field sprayed with these pesticides at the rate recommended for a vegetable crop. In the laboratory, the neutral red retention time (NRRT) was significantly reduced following exposure to both pesticides compared with controls. In the semi-field experiment, earthworm NRRT was significantly reduced by both pesticides. These experiments have shown that the NRRA is very sensitive to exposure to chlorpyrifos and diazinon even at field rates. It therefore shows promise as a potential biomarker of contamination of soil by organophosphates.     

N2O-producing microorganisms in the gut of the earthworm Aporrectodea caliginosa are indicative of ingested soil bacteria

The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N(2)O) and (ii) to characterize the N(2)O-producing bacteria of the earthworm gut. The production of N(2)O in the gut of garden soil earthworms (Aporrectodea caliginosa) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N(2)O were transient products when supplemental nitrate was reduced to N(2) by gut content homogenates. In contrast, nitrite and N(2)O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N(2)O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N(2)O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G+C contents, and 5 isolates were denitrifiers and reduced nitrate to N(2)O or N(2). The initial N(2)O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N(2)O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N(2)O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.     

Sublethal effects of imidacloprid on Bemisia tabaci (Hemiptera: Aleyrodidae) under laboratory conditions

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most important pests in tropical and subtropical agriculture and is a key pest in greenhouse production worldwide. Current management of B. tabaci relies upon frequent applications of insecticides. Insecticide use not only directly affects pest populations through acute toxicity but also has indirect (sublethal) effects on pest physiology or behavior. In this study, we described sublethal effects of imidacloprid on adult feeding, immature development, adult fecundity, and F1 development of B. tabaci. Honeydew excretion of adults feeding on leaves treated with LC20 and LC40 concentration was significantly lower than that on untreated leaf discs. Egg production of B. tabaci adults subject to LC20 and LC40 concentrations also was less than untreated individuals. Upon transfer to untreated leaves, honeydew excretion and egg production recovered well within 24 and 48 h, respectively. Exposure to LC20 and LC40 concentrations significantly affected developmental time of B. tabaci eggs and nymphs, whereas it did not affect adult molting rate. We did not find sublethal effects on longevity and fecundity of B. tabaci adults when exposed to LC90 and LC40 concentrations for 24 h, and on egg hatching rate, nymphal mortality, and molting rate of the subsequent F1 generation. Exposure to imidacloprid at LC40 concentration significantly decreased the number of females in the F1 generation. Imidacloprid negatively affects development and reproduction of exposed individuals, and sex ratio of subsequent (F1) generation of B. tabaci, which probably disrupts B. tabaci population dynamics, slows population increase, and reduces infestation levels. Therefore, it is necessary to consider potential impact from imidacloprid for integrated management of the pest.     

Effect of the Earthworms Lumbricus terrestris and Aporrectodea caliginosa on Bacterial Diversity in Soil

Earthworms ingest large amounts of soil and have the potential to radically alter the biomass, activity, and structure of the soil microbial community. In this study, the diversity of eight bacterial groups from fresh soil, gut, and casts of the earthworms Lumbricus terrestris and Aporrectodea caliginosa were studied by single-strand conformation polymorphism (SSCP) analysis using both newly designed 16S rRNA gene-specific primer sets targeting Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, and Firmicutes and a conventional universal primer set for SSCP, with RNA and DNA as templates. In parallel, the study of the relative abundance of these taxonomic groups in the same samples was performed using fluorescence in situ hybridization. Bacteroidetes, Alphaproteobacteria, and Betaproteobacteria were predominant in communities from the soil and worm cast samples. Representatives of classes Flavobacteria and Sphingobacteria (Bacteroidetes) and Pseudomonas spp. (low-abundant Gammaproteobacteria) were detected in soil and worm cast samples with conventional and taxon-targeting SSCP and through the sequence analysis of 16S rRNA clone libraries. Physiologically active unclassified Sphingomonadaceae (Alphaproteobacteria) and Alcaligenes spp. (Betaproteobacteria) also maintained their diversities during transit through the earthworm intestine and were found on taxon-targeting SSCP profiles from the soil and worm cast samples. In conclusion, our results suggest that some specific bacterial taxonomic groups maintain their diversity and even increase their relative numbers during transit through the gastrointestinal tract of earthworms.     

Stability of spinosad resistance in Frankliniella occidentalis (Pergande) under laboratory conditions

The stability of spinosad resistance in western flower thrips (WFT), Frankliniella occidentalis (Pergande), populations with differing initial frequencies of resistance was studied in laboratory conditions. The stability of resistance was assessed in bimonthly residual bioassays in five populations with initial frequencies of 100, 75, 50, 25 and 0% of resistant individuals. There were no consistent changes in susceptibility of the susceptible strain after eight months without insecticide pressure. In the resistant strain, very highly resistant to spinosad (RF50>23,000-fold), resistance was maintained up to eight months without further exposure to spinosad. In the absence of any immigration of susceptible genes into the population, resistance was stable. In the case of the population with different initial frequency of resistant thrips, spinosad resistance declined significantly two months later in the absence of selection pressure. With successive generations, these strains did not change significantly in sensitivity. Spinosad resistance in F. occidentalis declined significantly in the absence of selection pressure and the presence of susceptible WFT. These results suggest that spinosad resistance probably is unstable under field conditions, primarily due to the immigration of susceptible WFT. Factors influencing stability or reversion of spinosad resistance are discussed.     

The effect of environmental parameters on growth,cholinesterase activity and glutathione S-transferase activity in the earthworm (Apporectodea caliginosa)

The effect of age and environmental parameters on growth, cholinesterase (ChE), and glutathione S-transferase (GST) activities were assessed in juvenile Apporectodea caliginosa earthworms. Earthworms were maintained in three types of soil- loam, sandy, and clay- at a range of moisture contents (15-30%), and temperatures (5-20 C). Earthworm age (1-3 months) had no significant affect on ChE activity. Growth rates were influenced by all environmental parameters tested and these effects were interrelated. Optimum conditions for growth appeared to be in loam or sandy soil with 25-30% moisture at 10-15 C. The GST activity was influenced by soil temperature and activity was significantly higher at 15 C than at 5 C and 10 C. Soil type also influenced GST activity and this influence was dependent on moisture content. In sandy soil GST activity was significantly lower at 30% moisture than at lower moisture contents, in loam soil GST activity was significantly higher at 15% moisture than at higher moisture contents, while in clay soil GST activity was not affected by moisture content. These results indicate that in field experiments when evaluating GST activity soil temperature and soil type need to be consistent between control and 'contaminated sites'. ChE activity was only affected by temperature, so this should be considered when comparing control and treatedareas.     

The effect of environmental parameters on growth, cholinesterase activity and glutathione S-transferase activity in the earthworm (Apporectodea caliginosa)

The effect of age and environmental parameters on growth, cholinesterase (ChE), and glutathione S-transferase (GST) activities were assessed in juvenile Apporectodea caliginosa earthworms. Earthworms were maintained in three types of soil- loam, sandy, and clay- at a range of moisture contents (15-30%), and temperatures (5-20 C). Earthworm age (1-3 months) had no significant affect on ChE activity. Growth rates were influenced by all environmental parameters tested and these effects were interrelated. Optimum conditions for growth appeared to be in loam or sandy soil with 25-30% moisture at 10-15 C. The GST activity was influenced by soil temperature and activity was significantly higher at 15 C than at 5 C and 10 C. Soil type also influenced GST activity and this influence was dependent on moisture content. In sandy soil GST activity was significantly lower at 30% moisture than at lower moisture contents, in loam soil GST activity was significantly higher at 15% moisture than at higher moisture contents, while in clay soil GST activity was not affected by moisture content. These results indicate that in field experiments when evaluating GST activity soil temperature and soil type need to be consistent between control and 'contaminated sites'. ChE activity was only affected by temperature, so this should be considered when comparing control and treatedareas.     

Survival of shelled acanthors of Moniliformis moniliformis (Acanthocephala) under laboratory conditions

Shelled acanthors (= eggs) of two isolates of Moniliformis moniliformis (Acanthocephala) were tested for their capacity to respond to a hatching stimulus in vitro and for their retention of infectivity to a natural intermediate host (Periplaneta americana). The shelled acanthors were stored for more than 120 weeks (Australian isolate) and for 104 weeks (Texan isolate) together with rat faeces in an incubator maintained at 22.2 +/- 0.1 degrees C. In both cases, infectivity to P. americana was shown to have been retained. In vitro tests of the hatching response were carried out on many occasions during this period of faecal storage. Shelled acanthors continued to respond and no differences were detected either between isolates or within an isolate through time.     

Reproduction of wild-caught marmosets (Saguinus labiatus labiatus) under laboratory conditions.

The reproductive performance of marmosets (Saguinus labiatus labiatus) was evaluated under laboratory conditions. Nineteen of 22 (86%) male-female pairs produced 32 live births during the first 18 months in the laboratory. The reproductive performance and survival rates were comparable to those of other Saguinus species maintained under similar conditions.     

Population dynamics of Monochamus carolinensis (Col., Cerambycidae) under laboratory conditions

The study of life history dynamics of the pine sawyer beetle Monochamus carolinensis (Olivier) is of interest because the beetles are vectors for the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, the causal agent of pine wilt in susceptible pine trees. Previous observation of a laboratory colony of M. carolinensis suggested numbers of adult beetles reared from logs varied little in relation to oviposition densities among individual logs. Logs in laboratory colonies are routinely subjected to artificially high oviposition rates. The objectives of this study were to examine the relationship between initial oviposition rate, holding time (time between log cut and oviposition), log volume and area and adult survivorship in laboratory colony. This study examined the number of eggs, larvae, adults and generation survivorship in logs over a 2‐year period. The number of adults emerged per log was not related to the number of eggs laid. Late larval and adult numbers were significantly correlated with log size. Only 12% of the initial cohort completed development and emerged as adults. We attribute the high within‐log mortality of beetles to intraspecific competition and cannibalism.     

Larval development of Hypsophrys nicaraguensis (Pisces: Cichlidae) under laboratory conditions

The cichlid Hypsophrys nicaraguensis is a popular fish known as butterfly, and despite its widespread use as pets, little is known about its reproductive biology. In order to contribute to this knowledge, the study describes the relevant larval development characteristics, from adult and larval cultures in captivity. Every 12h, samples of larvae were collected and observed under the microscope for larval stage development, and every 24h morphometric measurements were taken. Observations showed that at 120h, some larvae had swimming activity and the pectoral fins development was visible; at 144h, the dorsal fin appear and all larvae started food intake; at 168h, the formation of anal fins begins, small rudiments of pelvic fins emerge, the separation of caudal fin from anal and dorsal fins starts, and the yolk sac is reabsorbed almost completely; at 288h, the pelvic fins starts to form; at 432h, the rays and spines of dorsal and anal fins can be distinguished, both the anal and the dorsal fins have the same number of spines and rays as in adults. After 480h larvae have the first scales, ending the larval stages and starting the transformation to fingerlings. Larvae were successfully fed with commercial diet.     

Reproduction of the greater bushbaby (Galago crassicaudatus panganiensis) under laboratory conditions

A breeding colony of Galago crassicaudatus panganiensis was maintained under laboratory conditions for four years, August 1971 to July 1975. The estrous cycle and the gestation period averaged 39 and 132 days in length, respectively. Twinning occurred in 14.5% of the pregnancies. No postpartum estrus was observed, although 1.2 conceptions per female per year were recorded. Breeding occurred throughout the year. Puberty occurred between ten and 18 months of age, the first conception occurred between 15 and 18 months of age, and reproductive capability decreased rapidly between ten and 12 years of age.     

Reproductive traits of Monochamus galloprovincialis (Coleoptera: Cerambycidae) under laboratory conditions

The pine sawyer Monochamus galloprovincialis (Olivier) is the vector of the introduced pine wood nematode Bursaphelenchus xylophilus (Steiner & Bührer) Nickle in Portugal, and until recently was considered a secondary forest insect. Under laboratory conditions, a study of biological and reproductive traits of 37 insect pairs was conducted. The longevity of both sexes was similar, being 61.2+/-6.5 days for males and 64.0+/-6.3 days for females (mean+/-SE). Sixteen small-sized insects (22% of the population) died within 20 days and before starting to reproduce. The sexual maturation period (without egg laying) was 20.4+/-0.7 days (mean+/-SE), while the oviposition period lasted 54.0+/-4.2 days (mean+/-SE). The oviposition rate increased very quickly during the first weeks of life, peaking to almost two eggs per day during days 30-44, and gradually dropping in the following weeks. The females laid an average of 67.0+/-5.96 (mean+/-SE) eggs through their lives. The hatch rate was 92.6+/-1.0% (mean+/-SE). There were large individual variations in longevity and fecundity parameters, and principal component analysis based on 16 morphological and biological parameters separated the breeding insects into four distinct groups. Almost half of the reproducing beetles were large-sized insects, with high longevity and fecundity traits. Overall, the reproductive potential of the Portuguese population of M. galloprovincialis seems to be smaller than that described for other Monochamus vectors of the pine wood nematode both in North America and Japan.     

Predation of cockles (Cerastoderma edule) by the whelk (Buccinum undatum) under laboratory conditions

The feeding rate and behaviour of whelks (Buccinum undatum)offered cockles (Cerastoderma edule) in laboratory experimentswere examined. When presented with cockles in a range of sizes(10–40 mm), 14 B. undatum (34.6–88.3 mm),held individually in aquaria, consumed a wide size range ofcockles. Small whelks (<40 mm) consumed cockles (<23 mm),whereas large whelks, (>60 mm) ate a greater numberof larger cockles (>30 mm) and a wider size range ofcockles (12–40 mm) than smaller whelks. The majority(90%) of the shells of the predated cockles were undamaged andthe few (<10%) that were damaged showed only slight abrasionsto the anterior and posterior shell margin. Filmed observationsof B. undatum feeding on C. edule showed a method of attackthat has not previously been reported and involved the use ofthe whelk's foot to asphyxiate the cockle or to pull the shellvalves apart. No filmed evidence was found for the previouslyreported shell ‘wedging’ technique for prising openthe closed shell valves of C. edule, although 10% of the shellsof consumed cockles in feeding experiments had damaged shellmargins. (Received 4 April 2007; accepted 30 June 2007)     

Life table for Selenothrips rubrocinctus (Thripidae: Panchaetothripinae) under laboratory conditions

The tropical thrip Selenothrips rubrocinctus Giard is a polyphagous species sometimes found in commercially important crops that include cocoa and cotton. Laboratory data from specimens collected in Psidium friedrichsthalianum trees in Costa Rica were used to prepare a life table. There was a high mortality in the egg (72.5%) and larva II (18.4%) stages. On leaves, inmature stages presented subvascular distribution and pupae presented submarginal distribution.     

Biology of Dermacentor silvarum (Acari: Ixodidae) under laboratory conditions

The minimum life cycle of Dermacentor silvarum Olenev had a mean duration of 87.5 days (range 74–102 days) under laboratory conditions [(27±1 °C), 70% RH, 6 L: 18 D]. The mean time in (days) for the different stages of its cycle was as follows: incubation period of eggs was 15.3 days; prefeeding, feeding and premoulting periods of larvae and nymphs averaged 5.5, 4.0 and 7.3 days, and 5.2, 5.0 and 14.6 days, respectively; prefeeding, feeding, preoviposition and oviposition periods of female adults lasted for 7.8, 4.5, 4.3 and 14.0 days, respectively. There existed a highly significant correlation between engorged body weight of females and egg masses laid (r = 0.9877, p<0.001). the= reproductive= efficiency= index= (rei)= and= reproductive= fitness= index= (rfi)= in= females= were= 11.09= and= 9.58,= respectively.= no= relationship= between= nymphal= engorged= body= weight= and= resultant= sexes= was= observed.= delayed= feeding= and= non-oviposition= (in= june= and= july)= existed= in= females,= and= low= temperature= (−10 °c)= treatment= for= 45 days= could= terminate= oviposition= diapause.= however,= the= egg= masses= laid= by= post-diapause= females= were= significantly= smaller= than= those= laid= by= females= engorged= in= march,= april= and=>