Hybridisations between Mytilus edulis and Mytilus galloprovincialis and performance of pure species and hybrid veliger larvae at different temperatures

The mussels Mytilus edulis L. and Mytilus galloprovincialis Lamark hybridise naturally in the wild along the Atlantic coast of Europe producing a patchwork of mixed pure species and hybrid populations. Individuals of both species were spawned in the laboratory and were hybridised in a series of reciprocal crosses. After 72 h, the proportion of eggs which developed into larvae (%yield) and the proportion of those larvae which had a normal veliger morphology (%normality) were estimated and compared between pure species and hybrid families. There were no significant differences in %yield or %normality between pure species and hybrids, but significant differences were evident between the offspring from different parents irrespective of whether they were hybrids or pure species. Therefore confirmation of hybrid heterosis in laboratory studies should not be based on a single, or a few reciprocal crosses. Hybrid and pure species veliger larvae were grown for approximately 4 weeks at 10, 14 or 20 °C. In all trials, pure M. galloprovincialis larvae grew significantly faster at 20 °C than either reciprocal hybrid or pure M. edulis larvae. Irrespective of temperature, in general, hybrid larvae grew slower than larvae of either pure species. Increased exposure to planktonic predation due to slow growth can be interpreted as selection against hybrids and this may play a role in the structure and distribution of mixed pure species and hybrid populations.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus

Phlebotomine sand flies are the only proven biological vectors of Leishmania parasites. However, Rhipicephalus sanguineus ticks have long been suspected to transmit Leishmania infantum in studies carried out in laboratory and natural conditions. In the present study, 5 μl of L. infantum promastigotes (1 × 10⁶ cells per ml) was injected into the hemocel through the coxa I of four engorged females (F1, F2, F3 and F4). Control ticks (F5 and F6) were injected with sterile phosphate-buffered saline (PBS) using the same procedure. Then, these females, their eggs, and the originated larvae were tested by real time polymerase chain reaction (real-time PCR) for the presence of L. infantum kinetoplast DNA (kDNA). Females and eggs were tested after the end of the oviposition period (about 5 weeks post-inoculation) whereas larvae were tested about 4 months after the inoculation of females. All artificially infected females were positive for L. infantum kDNA. In addition, two pools of eggs (one from F2 and other from F4) and four pools of larvae (one from each F1 and F4 and two from F2) were positive for L. infantum kDNA. These results showed, for the first time, the transovarial passage of L. infantum kDNA in R. sanguineus ticks, thus suggesting that the transovarial transmission of L. infantum protozoa in ticks is worth to be investigated.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

Thermal response and reversibility of prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis undergoes prolonged larval diapause that is terminated by chilling and warming cycles. To examine the effects of warming temperatures and their duration on diapause termination, we exposed diapause larvae that had not been reactivated after chilling at 5 °C to 20 or 25 °C and chilled them again before incubation at 20 °C. With increasing warming duration at 20 °C, diapause termination after chilling increased and shorter chilling durations became effective. In contrast, few or no larvae warmed at 25 °C terminated diapause after chilling, irrespective of the warming duration. To investigate the effect of warming temperature on diapause intensity, larvae with diapause weakened by initial incubation at 20 °C after the first chilling were subsequently incubated at 15, 20, or 25 °C, then chilled at 5 °C before incubation at 20 °C. Diapause termination increased significantly after the larvae were treated at 15 or 20 °C but decreased significantly after they were treated at 25 °C. The intensification of prolonged diapause at 25 °C was reversed when the larvae were transferred to 20 °C. Diapause intensity in C. sikkimensis therefore decreases at 20 °C, increases at 25 °C, and can be reversed by alternately exposing diapause larvae to 20 and 25 °C. In C. sikkimensis, prolonged diapause does not always proceed in one direction, and its intensity fluctuates in response to ambient temperature conditions.     

Extension of the breeding season and its effects on fertilization and development in two species of lugworm (Arenicola marina and A. defodiens)

In the autumn/winter breeding polychaetes, Arenicola marina and A. defodiens, spawning can be advanced or delayed by a number of months through temperature manipulation of the adults. However, this manipulation may have significant consequences for fertilization rates and embryo developmental success and so in vitro fertilizations were performed to assess the impact of manipulation. Firstly, we used oocytes and sperm obtained from advanced or delayed individuals. For both species, using gametes from 4 weeks advanced individuals did not result in a significant reduction in development, however, gametes from individuals advanced (A. marina only) or delayed by 8 weeks resulted in significantly fewer embryos developing normally. Reciprocal crosses of temperature-manipulated A. marina gametes (from 4 weeks advanced and 4 weeks delayed individuals) with those at the natural spawning time confirmed that the reduction in developmental success in both was attributable to the oocytes. After 5 h post-fertilization, the majority of oocytes from advanced individuals had fertilized, but by 24 h most were abnormal. For fertilizations with gametes from delayed individuals, nearly 100% of the embryos were developing normally after 24 h, but after 144 h significantly more were abnormal in crosses involving oocytes from delayed females. Although both species have reproductive plasticity to extend their breeding season, the significant reduction in the numbers of competent larvae produced as the spawning is delayed or advanced further may be a significant bottleneck in aquaculture and it may also have considerable implications for the long-term reproductive success of a population in response to environmental change.Sympatric populations of the species exist in many locations and the inherent variability in the breeding seasons could allow spawning times to overlap. Artificially delaying A. marina individuals enabled fertilizations to be performed with A. defodiens at the natural spawning time in the laboratory. Both conspecific fertilizations produced 100% trochophore larvae after 120 h, but A. defodiens oocytes failed to fertilize after incubation with A. marina sperm, in comparison to the A. marina oocytes incubated with A. defodiens sperm where 40% developed to the trochophore stage. This asymmetric gamete incompatibility may be one of a suite of mechanisms to minimise hybridisation.     

Benthic assemblages and temperature effects on Paracentrotus lividus and Arbacia lixula larvae and settlement

Recruitment is a principal controlling factor in population dynamics of marine species. In marine invertebrates with a planktonic larval stage, such as echinoids, recruitment is assured by larval supply, settlement and juvenile survival. Larval supply and juvenile survival are affected by a wide range of factors, including temperature, presence of predators, quality and quantity of food. Echinoid larval settlement is mainly conditioned by the finding of a suitable substrate to metamorphose. The sea urchins Arbacia lixula and Paracentrotus lividus are considered key species of the Mediterranean infralittoral rocky shores. At high densities, the grazing activity of both species can produce and maintain barren grounds, a particular habitat condition characterized by extremely low cover values of erect algae with high presence of naked substrates and encrusting corallinales, poor in biodiversity and ecosystem functions. We tested the role of different settlement substrates on the metamorphosis competent larvae of the two species. Furthermore, from our larval rearing trails we were able to identify strong temperature effects on larval development of the two species. P. lividus and A. lixula larvae have been reared at 18 °C but for the second species it was necessary to use higher temperatures (22 °C) to perform settlement experiments, as in the 18 °C set all larvae died in the first week. Both species larvae have been fed Cricosphaera elongata. Metamorphosis of competent larvae has been induced using different substrates: naked stones, Lithophyllum incrustans, Stypocaulon scoparium, Corallina elongata, turf forming algae and Posidonia oceanica. For each species, two larval batches were used for settlement experiments; for each larval batch two replicates/substrates were set up. No differences in the rate of metamorphosis on any of the tested substrates were observed for P. lividus, while A. lixula showed to prefer naked stones and encrusting coralline algae Considering that A. lixula population growth may trigger barren extension on rocky shores, this may lead to a positive feedback between barren extension and A. lixula population density. Furthermore, our results suggest that the predicted rise in seawater temperature may favor A. lixula larval survival and inhibit P. lividus. Combining information on temperature tolerance with other sources of information for these species in the Mediterranean, it is possible to develop a conceptual model of the interaction between the two species and the alternative state of their habitats.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Ontogenetic changes in the critical swimming speed of Gadus morhua (Atlantic cod) and Myoxocephalus scorpius (shorthorn sculpin) larvae and the role of temperature

Most studies on behavioural contributions to dispersal and recruitment during early life history stages of fishes have focused on coral reef species. For cold ocean environments, high variation in seasonal temperature and development times suggest that parallel studies on active behaviour are needed for cold-water species. Thus, we examined the critical swimming speed (U_crit) of marine fish larvae from 2 contrasting species: Gadus morhua (Atlantic cod) and Myoxocephalus scorpius (shorthorn sculpin), a pelagic and bottom spawner respectively. Within-species comparisons showed that sculpin reared at 6 °C had lower initial U_crit values, but a faster U_crit increase through development compared with 3 °C conspecifics, ultimately resulting in faster critical swimming speeds at metamorphosis (10.5 vs. 9.1 cm·s^− 1). In contrast, although cod larvae reared at 10 °C were faster swimmers at first feeding than 6 °C fish, temperature differences were absent after the first week. These results show that temperature influences the trajectory of larval critical swimming speed development, but that the relationship is species-specific. Although 6 °C sculpin and cod of similar length had equivalent U_crit values, the smaller size of cod at hatch (5.3 vs. 10.8 mm for sculpin) resulted in much lower age-specific U_crit values for cod. These data have significant implications for how swimming activity of the two species might affect dispersal, particularly in the first few weeks post-hatch. Overall, our data suggest that temperature during larval development influences the swimming capacity of cold-water marine fishes, and has important ramifications for biophysical models of dispersal.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

Distributional range extension of the seagrass Halophila nipponica into coastal waters off the Korean peninsula

Eight temperate seagrass species (five in the genus Zostera, two in the genus Phyllospadix, and Ruppia maritima) have been previously reported in coastal waters off the Korean peninsula, which lies between 33°N and 43°N. Recently, a species of Halophila, a genus which occurs predominantly in tropical and subtropical areas, has been observed on the southern coast of Korea for the first time. The species was identified as Halophila nipponica. H. nipponica is distributed in warm temperate regions of Japan influenced by the warm Tsushima Current and was previously unknown outside the Japanese archipelago. Thus, we are able to report a range extension into Korea. The Korean Halophila meadow that we observed covered an area of about 2.1 ha, with average shoot density of about 1300 m⁻². We measured morphological features of vegetative and reproductive organs between June and September 2007. Morphological and reproductive features of the Halophila species in Korea were similar to those of the species in Japan. Increased water temperature in the coastal waters of Korea may at least partially account for the persistence of this new population.     

The impact of food type, temperature and starvation on larval development of Balanus amphitrite Darwin (Cirripedia: Thoracica)

The impact of diatom food species (Chaetoceros calcitrans and Skeletonema costatum), temperature and starvation on the larval development of Balanus amphitrite was evaluated. Starvation threshold levels for different ages of larvae (0- to 5-day-old) fed with C. calcitrans and S. costatum and then starved at 5, 15 and 25 °C temperature were estimated as ultimate recovery hour (URH; denoting the starvation point in hours at the end of which larvae can recover and continue development). Effect of temperature on starvation threshold varied significantly with larval age and food species. The URH declined with larval age at 5 °C, but not at 15 and 25 °C. The URH and grazing rates were high for early instars fed on C. calcitrans, and for advanced instars fed on S. costatum. Carbon gain through feeding was maximum for 2-day-old larvae when fed with C. calcitrans and decreased with larval age. However, when fed with S. costatum carbon gain increased with larval age. This confirms that with development the utility of food types changes. The differences in the carbon gain can be attributed to differences in grazing rate due to variations in the size of the diatom cells, larval intersetular distance, diatom sinking rate and the photo-taxic behavior of larvae. Molting was observed at times when larvae were undergoing starvation and this could be viewed as stress-induced molting, and it differed with the larval age and food organisms.     

Metamorphosis in a semiterrestrial crab, Sesarma curacaoense: intra- and interspecific settlement cues from adult odors

The larvae of many marine invertebrate species are able to delay their settlement and metamorphosis in the absence of characteristic cues from the adult habitat. This phenomenon was experimentally studied in the megalopa stage of Sesarma curacaoense de Man, 1892, a semiterrestrial grapsid crab that lives in the shallow coastal mangrove habitats in the Caribbean region. Duration of the development and survival to metamorphosis to the first juvenile crab stage were compared between experimental treatments, where the water was conditioned with adult crabs (“adult-conditioned water,” ACW) and control groups reared in filtered seawater. In the experiments with larvae from two different females, development duration was significantly shorter and mortality lower in water conditioned with conspecific adults. In the two control groups, the effects of supply with an artificial substrate (nylon gauze) were tested. This comparison showed that the presence of substrate did not significantly influence the time to metamorphosis, but did reduce the mortality rate. In all later experiments, the megalopae were thus routinely provided with nylon gauze as a substrate. In each of the three subsequent replicate experiments conducted with larvae from different females, survival rate and development time to metamorphosis were compared between one control group and four treatments with ACW. The effectiveness of conspecific (S. curacaoense) adult odors as metamorphosis-stimulating cue was, in these experiments, compared with that of ACW from one congener (S. rectum) and two species belonging to different genera within the Grapsidae (Armases miersii, Chasmagnathus granulata). While the rate of survival showed inconsistent patterns among repeated experiments, the development was consistently fastest with conspecific ACW, followed by ACW from S. rectum, A. miersii and C. granulata. Only the conspecific and congeneric cues had statistically significant effects (i.e. shorter development than in the controls). These response patterns suggest that chemically similar factors (presumably pheromones) are produced by closely related species and, thus, their chemical structure may reflect phylogenetical relationships within a clade.     

Evaluation of real-time PCR targeting hbb gene for Borrelia species identification

Several molecular methods have been employed for Borrelia species identification. Newly developed technology, real-time polymerase chain reaction (RT-PCR), combines simultaneous amplification, detection and differentiation of strains in one PCR run. The aim of the study was to perform and evaluate RT-PCR for Borreliaburgdorferi sensu lato species identification. Borrelia species identification was accomplished on 374 Borrelia strains using two approaches: 1.) MluI restriction of entire borrelial chromosome (MluI-large restriction fragment patterns, LRFP), and 2.) RT-PCR targeting hbb gene and specific melting temperature (Tm) detection. The results of the two molecular methods were compared. With MluI-RFLP we were able to differentiate all Borrelia species and their subtypes within particular species. RT-PCR based on Tm determination identified unique strains within the species Borreliaafzelii (Tm 66.11 °C), B. burgdorferi sensu stricto (Tm 68.18 °C), Borreliaspielmanii (Tm 59.45 °C) and Borreliavalaisiana (Tm 59.62 °C). We were not able to distinguish the last two species that shared almost identical Tm. The large majority of Borreliagarinii strains shared Tm 51.42 °C, while subtype Mlg4 was characterized by Tm 56.87 °C. Strains of Borrelialusitaniae species also were heterogeneous; human isolate had Tm 63.47 °C while two tick isolates shared Tm 61.77 °C. Differences inside hbb gene enabled differentiation of the majority of Borrelia species, and revealed two clusters within B. garinii and B. lusitaniae species, respectively, but it was not possible to distinguish B. spielmanii form B. valaisiana. The major advantage of RT-PCR was that it was easy to perform and that the results were obtained within a few hours.     

Performance and thermal sensitivity of the southernmost lizards in the world, Liolaemus sarmientoi and Liolaemus magellanicus

Locomotor activity performance of reptiles is largely temperature dependent and, in harsh environments with short activity periods during the day and throughout the year, plays a vital role in the fitness of the species. This particular study focuses on the performance and the thermal sensitivity for running, at different body temperatures, of the two southernmost species of lizards in the world, Liolaemus sarmientoi and Liolaemus magellanicus, studied at two locations in the south of Santa Cruz province, Argentina (51°S, 70°W and 50°S, 72°W; 133 m asl). The speed of sprint and long runs was measured in the field to determine the physiological performance of lizards at different air temperatures. In both species speed increases with the temperature, and they reach the highest performance at high temperatures. The difference between activity and thermal optima suggests that L. magellanicus has colonized its actual environment recently, and that it has not had enough time for its physiological mechanisms to evolve and achieve a maximum performance at the cold temperatures they have to tolerate at present. In contrast, L. sarmientoi achieved a high performance over a wider range of temperatures that included temperatures lower than the preferred temperatures in the lab, which they can generally find in their environment.     

Larval and early juvenile development of Lithodes santolla (Molina, 1782) (Decapoda: Anomura: Lithodidae) reared at different temperatures in the laboratory

The southern king crab, Lithodes santolla Molina, is distributed in cold-temperate and subantarctic waters ranging from the southeastern Pacific island of Chiloé (Chile) and the deep Atlantic waters off Uruguay, south to the Beagle Channel (Tierra del Fuego, Argentina/Chile). Recent investigations have shown that its complete larval development from hatching to metamorphosis, comprising three zoeal stages and a megalopa, is fully lecithotrophic, i.e. independent of food. In the present study, larvae were individually reared in the laboratory at seven constant temperatures ranging from 1 to 18 °C, and rates of survival and development through successive larval and early juvenile stages were monitored throughout a period of 1 year. The highest temperature (18 °C) caused complete mortality within 1 week; only a single individual moulted under this condition, 2 days after hatching, to the second zoeal stage, while all other larvae died later in the zoea I stage. At the coldest condition (1 °C), 71% of the larvae reached the zoea III stage, but none of these moulted successfully to a megalopa. A temperature of 3 °C allowed for some survival to the megalopa stage (17-33% in larvae obtained from two different females), but only a single individual passed successfully, 129 days after hatching, through metamorphosis to the first juvenile crab instar. At all other experimental conditions (6, 9, 12 and 15 °C), survival through metamorphosis varied among temperatures and two hatches from 29% to 90% without showing a consistent trend. The time of nonfeeding development from hatching to metamorphosis lasted, on average, from 19 days at 15 °C to 65 days at 6 °C. The relationship between the time of development through individual larval or juvenile stages (D) and temperature (T) was described as a power function (D=aT^b, or log[D]=log[a]blog[T]). The same model was also used to describe the temperature dependence of cumulative periods of development from hatching to later larval or juvenile stages. One year after hatching, the 7th (6 °C) to 9th (15 °C) crab instar was reached. Under natural temperature conditions in the region of origin of our material (Beagle Channel, Argentina), L. santolla should reach metamorphosis in October-December, i.e. ca. 2 months after hatching (taking place in winter and early spring). Within 1 year from hatching, the crabs should grow approximately to juvenile instars VII-VIII. Our results indicate that the early life-history stages of L. santolla tolerate moderate cold stress as well as planktonic food-limitation in winter, implying that this species is well adapted to subantarctic environments with low temperatures and a short seasonal plankton production.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Laboratory evaluation of the chemosterilant lufenuron against the fruit flies Ceratitis capitata, Bactrocera dorsalis, B. cucurbitae, and B. latifrons

Four species of tephritid fruit flies, Ceratitis capitata, Bactrocera dorsalis, B. cucurbitae, and B. latifrons were evaluated for toxic, developmental, and physiological responses to the chemosterilant lufenuron. No significant mortality of laboratory strains of the first three species was observed after their exposure up to 50 μg/mL of lufenuron in agar adult diet, whereas B. latifrons adults fed with 50 μg/mL of lufenuron in the diet caused significant mortality compared to the control. Fertility of C. capitata adults fed on 50 μg/mL lufenuron-fortified diet between 7 and 12 days of age was approximately 46% of the no lufenuron control. Fertility of B. dorsalis and B. latifrons adults fed on 50 μg/mL lufenuron-incorporated diet was about 45% and 62% of the control, respectively. Lufenuron did not significantly affect fertility of B. cucurbitae adults. Lufenuron did not affect fecundity of C. capitata and B. dorsalis. Fecundity of B. cucurbitae and B. latifrons was not evaluated due to difficulty to count the eggs laid deep in the agar diet. Larvae fed on a liquid larval diet with ≤ 0.1 μg/mL of lufenuron were also evaluated. Pupal recovery, adult emergence, adult fliers, mating, egg hatch, and egg production of C. capitata were significantly decreased, while for B. dorsalis, pupal recovery, larval duration and adult emergence were affected. No effect of lufenuron on B. cucurbitae larvae was observed. B. latifrons was not performed because shortage of eggs at the time of this research. Lufenuron is a potential agent for management and control of C. capitata and B. dorsalis.     

Further research on the production, longevity and infectivity of the zoospores of Leptolegnia chapmanii Seymour (Oomycota: Peronosporomycetes)

The effect of temperature on the production, survival and infectivity of zoospores of an Argentinean isolate of Leptolegnia chapmanii was determined under laboratory conditions. Production of zoospores of L. chapmaniiin vitro and in vivo upon first and fourth instars larvae of the mosquito Aedes aegypti was studied at three different temperatures. Zoospores from infected larvae were infective to mosquito larvae for 51, 12, and 5 consecutive days when maintained at 25, 35, and 10 °C, respectively. Maximum zoospore production in infected fourth-instar larvae was 9.6 ± 1.4 × 10⁴ zoosp/larva at 48 h at 25 °C. The average number of zoospores produced by individual fourth-instar Ae. aegypti larvae infected with L. chapmanii was 3.57 ± 0.46 × 10⁵ zoospores during 6 consecutive days at 25 °C. Zoospore production in vitro was also affected by temperature with a maximum of zoospores (n = 47,666/ml) produced at 25 °C. When zoospores produced in vitro were used as inoculum against Ae. aegypti larvae at 25 °C, larval mortality was recorded for 5 consecutive weeks. The encystment process for zoospores took 17-20 min; the germination of cysts (excystment) occurred 5 min after exposure in water to mosquito larvae. The minimal time of contact between zoospores and mosquito larvae to develop infection was two minutes. Infection took place by zoospore attachment onto and then penetration through the larval cuticle or by ingestion of cysts as was confirmed by histological studies. Temperature directly affected infectivity and production of zoospores in vivo and in vitro although L. chapmanii zoospores tolerate a wide range of temperatures.