Nosema takapauensis n.sp., a microsporidan parasite of larvae of Costelytra zealandica (Coleoptera: Scarabaeidae) in New Zealand

Morphological and biological features of a microsporidan protozoan parasite of larvae of Costelytra zealandica collected at Takapau, southern Hawkes Bay, are described and evaluated taxonomically. The parasite multiplies in the fat body of all larval instars, causing massive tissue disintegration in advanced infection resulting in the retardation of development and ultimately death before pupation. The microsporidan forms one spore per sporont, and therefore belongs to genus Nosema’, it is considered to be specifically distinct from its nearest congener, N. melolonthae.     

Influence of low temperature on pupation induction in Costelytra zealandica (Coleoptera: Scarabaeidae)

Costelytra zealandica (White) lives within the soil throughout its life cycle, except for brief, crepuscular excursions to the surface by the adults to mate and feed. Larvae collected more than 18 weeks before 50% field pupation did not pupate unless they were artificially chilled. Such chilling had no significant effect on the percentage pupation of larvae collected after this time; it is assumed that these larvae had received sufficient chilling from their environment to induce pupation. Larger larvae were more likely to pupate than smaller larvae during the period 13–7 weeks before 50% field pupation. It is postulated that larvae remain quiescent until the soil temperature increases in the spring, and pupate only then. Such a process would explain how the adults of a population emerge at about the same time.     

A correlated response of a parasite’s virulence and life cycle to selection on its host’s life history

We demonstrate a correlated response of the virulence and the mode of transmission of the microsporidian parasite Edhazardia aedis to selection on the age at pupation of its host, the mosquito Aedes aegypti. We selected three lines of mosquitoes each for early or late pupation and exposed the larvae after zero, two and four generations of selection to a low and a high concentration of the parasite’s spores. Before selection the parasites induced a similar level of mortality in the six lines; after four generations of selection mortality was higher in the mosquitoes selected for late pupation than in those selected for early pupation. Overall, parasite-induced mortality was positively correlated with the mean age at pupation of the matching uninfected line. When they died, mosquitoes selected for early pupation harboured mostly binucleate spores, which are responsible for vertical transmission. Mosquitoes selected for late pupation were more likely to harbour uninucleate spores, which are responsible for horizontal transmission. The parasite enhanced this tendency for horizontal transmission by prolonging the larval period in the lines selected for late pupation, but not in the ones selected for early pupation. These results suggest that the genetic basis of the mosquito’s age at pupation helps to determine the parasite’s mode of transmission: parasites in rapidly developing mosquitoes are benign and transmit vertically, while parasites in slowly developing mosquitoes are virulent and transmit horizontally. Thus, as the host’s life history evolves, the parasite’s performance changes, because the host’s evolution changes the environment in which the parasite develops.     

Influence of temperature on developmental parameters of the parasite/host system Edhazardia aedis (Microsporida: Amblyosporidae) and Aedes aegypti (Diptera: Culicidae).

Larvae of Aedes aegypti, transovarially infected with Edhazardia aedis, were reared between 20 and 36 degrees C to determine the influence of temperature on the development of the parasite and the infected host. Development of the parasite was evaluated based on spore yield and size. The predicted optimum temperature for maximum spore production of E. aedis in A. aegypti was 30.8 degrees C. The results demonstrate that the E. aedis-A. aegypti system has a wide temperature tolerance; whereas spore yield will be lower at unfavorable temperatures, the host will remain infected. Additionally, spores were significantly smaller from individual reared at 34 degrees C than those reared at either 20 or 27 degrees C. Development of the infected host was evaluated based on pupal weight and time of pupation. Infected pupae were significantly larger than uninfected pupae. There was also a significant difference in the pupation rate between controls and infected A. aegypti larvae. Controls had a 50% cumulative pupation time (CPT50) of 65.7 degree days and infected individuals a CPT50 of 76.6 degree days.     

Biology of Microplitis kewleyi (Hymenoptera: Braconidae): A parasite of Agrotis ipsilon (Lepidoptera: Noctuidae) larvae

Microplitis kewleyi Muesebeck is a gregarious internal parasite of larvae of the black cutworm Agrotis ipsilon (Hufnagel). Studies of the biology of the parasite revealed that there was an inverse relationship between host instar and parasite preference. Duration of development from egg to pupa ranged from 18 days at 27°C to 68.7 days at 16°C. Development from egg to pupa took 13.5–21.6 days when fourth and first instar host larvae, respectively, were parasitized. A larger number of parasites emerged from hosts parasitized in the fourth instar (22.4) than the first instar (11.5). Parasite pupation occurred when the host was in the fifth/sixth instar, depending on the instar parasitized. Thirty‐nine per cent of host larvae exposed as first instars to parasites died before parasite emergence. This decreased to 0% for host larvae exposed as fourth instars. The sex ratio was 1:1.2 (M:F). Thirty‐seven per cent of hosts exposed diurnally were stung, compared to 24% exposed nocturnally. Mean daily progeny was highest (12) on the first day, decreasing to zero after 20 days. Percent host parasitism was also highest on the first day (35%) decreasing to nearly 0% after 18 days. There appear to be three parasite larval instars. Host larvae often remained alive after parasite emergence.     

Population biology of the broad-horned flour beetle,Gnathocerus cornutus (F.) II. Crowding effects of larvae on their survival and development

Crowding effects of larvae on survival and development were examined for the broad-horned flour beetle, Gnathocerus cornutus (F.). The larvae matured about 3 weeks after hatching regardless of their densities, but pupation was severely hindered by crowding. There existed an upper limit for the number of the pupae produced and its mechanism was studied by a statistical analysis of the distribution patterns of pupal cells and the experiment in which glass tubes were artificially supplied in addition as pupation site. These studies show that G. cornutus larvae have a habit to construct cells for pupation and this habit leads to a contest competition for pupation site at high densities. The significance of the contest competition for population regulation was discussed comparing the results on Tribolium confusumJacqueline duVal .     

Interaction between the tachinid parasite,Sturmiopsis inferens and granulosis virus in the sugarcane shoot borer,Chilo infuscatellus [Lep.: Crambidae]

Competition between granulosis virus (GV) and the larval parasite,Sturmiopsis inferens Tns. (Tachinidae: Diptera), was studied in 3^rd — and 4^th — instar larvae of the sugarcane shoot borer,Chilo infuscatellus Snellen (Crambidae: Lepidoptera), under laboratory conditions. Mortality due to GV infection and parasitization was 76.8 and 47.6 per cent, respectively, when they were tested separately. But when hosts were infected simultaneously with microfeeding of GV and larval parasite, a significantly low parasitism (5.5%) was obtained compared to 74.8 per cent mortality by GV infection. When the larvae were microfed with the GV 6 days after inoculation with parasitic maggots, mortality due to the virus was reduced significantly to 20.5 per cent, but when the maggot inoculation was preceded by virus microfeeding 6 days before, parasitization was unsuccessful, while 75% of larvae died of virus. Results obtained from field — collected larvae also showed that significantly more parasite puparia were recovered from healthy larvae than from virus — infected larvae. Similar differences in parasitization were not obtained in the case of healthy or virus — infected pupae.      

Synthesis and Anti-juvenile Hormone Activity of 1-Citronellyl-5-substituted Imidazoles

A number of 1-citronellyl-5-substituted imidazoles were synthesized and bioassayed on the silkworm, Bombyx mori, in order to assess their anti-juvenile hormone activity. Most of the 1-citronellyl-5-substituted imidazoles induced precocious metamorphosis in the 3rd and 4th instar larvae of B. mori by topical application. The percentage of precocious metamorphosis correlated well with the dosage. Among the compounds tested, l-citronellyl-5-(2-chlorophenyl)imidazole (8) and the 2-methylphenyl analog 10 showed the highest activity. When compounds 8 and 10 were applied to the 4th instar larvae at 10 μg/larva, precocious pupation was induced in 100% without any lethal effects.     

Retardation of testis development in the armyworm,Pseudaletia separata,parasitized by the braconid wasp,Cotesia kariyai

Summary

In order to complete growth and development, the endoparasitoid wasp, Cotesia (=Apanteles) kariyai, inhibits pupation of its armyworm host, Pseudaletia (=Leucania) separata. In host larvae retardation of testis and spermatocyst development caused by the parasitoid was also observed. The agents causing the retardation were found in the ovaries and venom of the female adult parasitoid. When an unparasitized male host larva was artificially injected with calyx fluid obtained from ovaries together with venom, it showed the same degree of developmental retardation of testes and spermatocysts as in natural parasitization. Testes implanted in isolated abdomens of healthy larvae did not increase in size by ecdysteroid stimulation after exposure to calyx fluid plus venom. It is suggested that both symbiotic polydnavirus existing in calyx fluid and venom in the parasitoid, C. kariyai, are responsible for the parasitic retardation of the male reproductive organs in the host, P. separata.     

Food availability and parasite infection influence the correlated responses of life history traits to selection for age at pupation in the mosquito Aedes aegypti

We selected six lines of mosquito Aedes aegypti for earlier or later pupation and measured the correlated responses of several life history traits: adult size, two fecundity measures and pre-adult survival. We further examined the influence of two environmental parameters – larval food availability and infection by the microsporidian parasite Edhazardia aedis– on the correlated responses. Pre-adult survival did not respond to selection for age at pupation in any environment. For all of the other traits, the environment influenced the correlated response, though the contribution of the different environmental aspects differed among traits. While the correlated response of adult size was influenced only by larval food availability, the likelihood that a female laid eggs was influenced by parasite infection, and the correlated response of the number of eggs was influenced by the interaction of the two environmental parameters. Generally, a deteriorating environment moved the correlated response from one favouring later pupation to one favouring earlier pupation. Larval food availability and parasite infection also influenced the association between the mean wing length and fecundity of the selected lines. At high food availability, there was a positive relationship between adult size and fecundity, while infected mosquitoes reared at low food availability showed the opposite trend. We discuss these results in light of the coevolutionary potential of the host–parasite interaction.     

Prothoracicotropic hormone bioassay: Bombyx larva assay

Summary

An assay for the prothoracicotropic hormone (PTTH) has been established using in situ activation of the prothoracic glands (PG) of Bombyx mori in its larva-to-larva development. The timing of PTTH release was estimated by examining developmental response of 4th instar larvae to brain removal and neck ligation, and changes in the haemolymph ecdysteroid titer and ecdysone-releasing activity of PG in vitro during the development. Injection of Bombyx brain extracts into 4th instar larvae neck-ligated shortly before full activation of PG elicited larval moulting rather than precocious pupation in headless larvae. This developmental shift was regarded as due to the action of PTTH, and the PTTH unit has been defined from a linear log dose-response relationship. Materials chromatographically fractionated from Bombyx brain extracts were examined for the presence of stage- and species-specific PTTH molecules by using this Bombyx larva assay and Bombyx and Samia pupa assays previously developed. The same fractions were active when assayed by Bombyx larva and pupa assays.     

Regulation of development of the grain beetle Tenebrio molitor by neuropeptides

Summary

Two neuropeptides (NPs), hydra head activator (HHA), and the luteinizing hormone releasing hormone (LHRH), were used to study their effects on the development of the larvae of the grain beetle Tenebrio molitor (mealworms). The larvae received the NPs in the course of development at the beginning or at the end of instar. The LHRH given at the beginning of the instar stimulated molting and pupation in larvae; it did not affect molting in the late period of the instar. The C-end fragment of LHRH_(9–10) produced fewer molts and increased the duration of the instar period. The effect of this fragment was weak when it was injected at the initial period of the instar; it was considerably stronger in the end of the instar. It inhibited development when administered during the last instar. On the contrary, the N-end fragment of the LHRH_(1–2) stimulated growth and development of larvae. The action of these NPs can be connected to the function of the hormones of metamorphosis. The HHA administrated to the larvae at different days of the instar stimulated molting but in the last instar it delayed pupation. The C-end of the HHA_(7–11) stimulated molting when injected during the whole period of the instar. The other C-end fragments, the HHA_(8–11) and HHA_(9–11) had inhibitory effects on the molting. The injection of the N-end fragment of the HHA_(1–5) had no effect. The action of HHA and its fragments did not correlate with the function of the hormones of metamorphosis.     

Circannual pupation rhythm in the varied carpet beetle Anthrenus verbasci under different nutrient conditions

Anthrenus verbasci pupates in spring and the timing of pupation is controlled by a circannual rhythm. Although A. verbasci is considered to be a univoltine species in Japan, it is assumed that larval development in its natural habitats, including bird nests, varies with nutrient availability, and that the life cycle often takes two or more years to complete. In the present study, larval development and pupation times were compared under constant and outdoor conditions in larvae provided a diet of either high‐nutrient bonito powder or low‐nutrient pigeon feathers. Although a circannual pupation rhythm was observed irrespective of the diet used, larval development was slower on feathers than on bonito powder. The pupation times on feathers varied over three years or more under both constant and outdoor conditions. Under outdoor conditions, larvae grown on feathers needed three years to approach the weight gained within a year by larvae grown on bonito powder. It is considered that life cycle length in A. verbasci is often two years or more in nutritionally unstable natural habitats, and that this species has probably evolved a circannual rhythm as a seasonal adaptation to nutrient‐poor environments.     

DEVELOPMENT OF ENDOPHYTIC FRANKIA SPORANGIA IN FIELD- AND LABORATORY-GROWN NODULES OF COMPTONIA PEREGRINA AND MYRICA GALE

Field-collected nodules of Comptonia peregrina (L.) Coult. and Myrica gale L. (Myricaceae), infected by the nitrogen-fixing actinomycete Frankia sp., were of two types: those that lacked sporangia entirely, designated spore(-), and those that showed extensive sporangial development, designated spore(+). In spore(+) nodules of C. peregrina, sporangia began to develop after the differentiation of endophytic vesicles and the concomitant onset of nitrogenase activity. At the onset of sporangial differentiation, infected host cells appeared healthy. However, endophytic vesicles and host cell cytoplasm and nuclei began to senesce rapidly as sporangia developed. Staining of sectioned material with the fluorescent stain Calcofluor White suggested that vesicles, hyphae and young sporangia were enclosed within a host-derived encapsulation layer, but mature sporangia were no longer encapsulated. In both C. peregrina and M. gale, vesicles were more short-lived in spore(+) than in spore(-) nodules. Field-collected spore(+) M. gale nodules exhibited a pronounced seasonality of sporangial formation. Sporangia began to differentiate in June, after the formation of vesicles and became more prominent in late summer. Inter- and intraspecific cross-inoculations suggest that the ability to form sporangia in the symbiotic state is controlled by endophytic strain type rather than host genotype or host/endophyte combination. The host may, however, influence the number and seasonal appearance of sporangia formed.     

The effects of Plagiorchis noblei metacercariae on the development and survival of Aedes aegypti larvae in the laboratory

Plagiorchis noblei infections impair the survival and development of fourth instar Aedes aegypti larvae. Mortality during the larval and pupal stages reached 92%, and 60% of the emerging adults were malformed. The metacercariae interfere with pupation and the emergence of adults. Larvae and pupae that fail to transform to the next developmental stage within the normal time characteristically persist for extended periods, but invariably die without transforming. Whereas 82% of the control larvae gave rise to functional adults, only 4% of infected larvae managed to do so. Such effects may facilitate the transmission of the parasite.     

A new method for the production of sterile colonies of Lucilia sericata

Maggot debridement therapy (MDT) refers to the use of blowfly larvae to clean or debride an infected wound. Most commonly, larvae of Lucilia sericata (Meigen) (Diptera: Calliphoridae) are used, and are sterilized prior to use to ensure no further bacterial infections are introduced during treatment. Current methods sterilize eggs from laboratory‐reared blowfly colonies, after which sterile early second instar maggots can be provided to hospitals for use in treatment. Maggots not required for treatment are used for colony regeneration, in which sterility is not maintained. The ability to maintain sterility beyond this would allow further research into fly–bacteria interactions and the effects of different bacteria on the blowfly lifecycle. This study aimed to produce a colony of sterile adults, using current egg sterilization practice, but maintaining sterility through to pupation and emergence. The production of a sterile colony allows further research into the impact of bacteria on fly development and survival. Eggs were placed on a sterile food source within autoclaved plant tissue culture containers to allow growth under sterile conditions. Nutrient agar plating of sterilized and non‐sterilized eggs, larvae and adults (post‐emergence), as well as the pupation medium and feed source in nutrient broth confirmed the aerobic sterility of all samples involved. The lifecycle of L. sericata was successfully completed through pupation to emergence with no effects on lifespan or oviposition by the newly emerged, sterile adult colony.     

Host regulation effects on Heliothis virescens (F.) larvae inducd by teratocytes of Cardiochiles nigriceps Viereck (Lepidoptera,Noctuidae-Hymenoptera,Braconidae)

Teratocytes deriving from the serosal membrane of Cardiochiles nigriceps Viereck, obtained “in vitro” from embryos hatched on a semidefined medium, were injected at different numbers and in different developmental stages of nonparasitized Heliothis virescens (F.) last instar larvae. Host development was affected by teratocyte injections and the responses registered ranged from normal to complete inhibition of pupation, according to the number of teratocytes injected and the developmental stage of the larva at time of injection. Complete pupation failure was observed when teratocytes derived from 4C nigriceps embryos were injected into 1st day 5th instar (new-slender stage) host larvae. Complete pupation occurred when teratocytes from 2 embryos were injected into 3rd or 4th day 5th instars (burrow-digging or day 1 cell formation stage). Intermediate responses, such as the formation of pupal cuticle without ecdysis or with only partial ecdysis, were obtained with intermediate teratocyte numbers, or host developmental stages. All pupae derived from teratocyte injected larvae failed to develop into adults normally obtained from control injected larvae. The larval weight just before pupation was negatively affected only when teratocyte injections were performed on 1st day 5th instar H. virescens larvae. Teratocyte injections altered the hemolymph protein titer to a level similar to that occurring in parasitized larvae. At the same time the ecdysteroid titer was characterized by a late significant increase, which reached values almost 3 times greater than found in normally parasitized larvae, and also surpassed the highest values registered for nonparasitized larvae. Ligation of parasitized larvae between the meso- and metathorax demonstrated that when the prothoracic glands were excluded, there was almost no ecdysteroid production posterior to the ligation. Ligations performed on parasitized larvae to isolate parasitoid eggs before hatching in the last abdominal segments, demonstrated that only virus and venom determined a reduction of the ecdysteroid titer. On the basis of these results the possible role of teratocytes in affecting the biological activity of ecdysteroids is postulated and discussed in a wider context of host-parasitoid physiological interactions.     

Genetic manipulation allows in vivo tracking of the life cycle of the son-killer symbiont,Arsenophonus nasoniae,and reveals patterns of host invasion,tropism and pathology

Maternally heritable symbionts are common in arthropods and represent important partners and antagonists. A major impediment to understanding the mechanistic basis of these symbioses has been lack of genetic manipulation tools, for instance, those enabling transgenic GFP expression systems for in vivo visualization. Here, we transform the ‘son-killer’ reproductive parasite Arsenophonus nasoniae that infects the parasitic wasp Nasonia vitripennis with the plasmid pOM1-gfp, re-introduce this strain to N. vitripennis and then used this system to track symbiont life history in vivo. These data revealed transfer of the symbiont into the fly pupa by N. vitripennis during oviposition and N. vitripennis larvae developing infection over time through feeding. A strong tropism of A. nasoniae to the N. vitripennis ovipositor developed during wasp pupation, which aids onward transmission. The symbiont was also visualized in diapause larvae. Occasional necrotic diapause larvae were observed which displayed intense systemic infection alongside widespread melanotic nodules indicative of an active but failed immune response. Our results provide the foundation for the study of this symbiosis through in vivo tracking of the fate of symbionts through host development, which is rarely achieved in heritable microbe/insect interactions.     

Effect of dietary selenium supplementation on resistance to baculovirus infection

We have examined the effects of dietary selenium (Se) supplementation on larval growth and immunocompetence of the lepidopteran pest, the cabbage looper, Trichoplusia ni. Supplementation of the diet of T. ni larvae with 10–20 ppm Se resulted in a 1 day delay in pupation. The effects of the addition and/or removal of dietary Se on total Se bioaccumulation and sequestration were determined by neutron activation analysis of pupae. Early penultimate instar larvae moved from selenium containing diet to basal diet lost total pupal Se content down to the level of those fed basal diet. Conversely, larvae moved from basal diet to diet containing additional Se rapidly attained pupal Se levels comparable to larvae fed Se throughout larval development. Therefore, dietary Se is rapidly accumulated or lost during larval development, but significant amounts are sequestered from diet into pupae. Larvae were reared on diet supplemented with 5 or 10 ppm Se until the onset of the penultimate instar then infected per os with increasing concentrations of the fatal baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Larvae fed Se in the penultimate and ultimate instars were more resistant to viral infection than larvae not fed Se in the final instars. This study indicates that dietary Se levels rapidly impact Se assimilation and sequestration and that tissue Se levels are an important factor in resistance to AcMNPV infection in larval T. ni.