Nonparasitized Larvae Research Articles

Transcriptomic Insights into Host Metabolism and Immunity Changes after Parasitization by Leptopilina myrica.

Parasitoids commonly manipulate their host's metabolism and immunity to facilitate their offspring survival, but the mechanisms remain poorly understood. Here, we deconstructed the manipulation strategy of a newly discovered parasitoid wasp, L. myrica, which parasitizes D. melanogaster. Using RNA-seq, we analyzed transcriptomes of L. myrica-parasitized and non-parasitized Drosophila host larvae. A total of 22.29 Gb and 23.85 Gb of clean reads were obtained from the two samples, respectively, and differential expression analysis identified 445 DEGs. Of them, 304 genes were upregulated and 141 genes were downregulated in parasitized hosts compared with non-parasitized larvae. Based on the functional annotations in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, we found that the genes involved in host nutrition metabolism were significantly upregulated, particularly in carbohydrate, amino acid, and lipid metabolism. We also identified 30 other metabolism-related DEGs, including hexokinase, fatty acid synthase, and UDP-glycosyltransferase (Ugt) genes. We observed that five Bomanin genes (Boms) and six antimicrobial peptides (AMPs) were upregulated. Moreover, a qRT-PCR analysis of 12 randomly selected DEGs confirmed the reproducibility and accuracy of the RNA-seq data. Our results provide a comprehensive transcriptomic analysis of how L. myrica manipulates its host, laying a solid foundation for studies on the regulatory mechanisms employed by parasitoid wasps in their hosts.

Adaptation and establishment of Habrobracon hebetor Say in the population of stored moths pests of date, Ephestia kuehniella Zeller and Plodia interpunctella Hübner

BackgroundHabrobracon hebetor (Hymenoptera: Braconidae) is a larval ectoparasitoid, mainly of pyralid moths of the stored pests of date, Plodia interpunctella Hübner and Ephestia kuehniella Zeller. It has some suitable characteristics for use in biological control of stored moth pests. The aim of this study was to compare the ability of H. hebetor establishment in the population of two important date storage moth pests P. interpunctella and E. kuehniella, and its role in their population fluctuations.ResultsThe population growth trends of two host species increased in the first 6 weeks and in the 17th to 22nd weeks of the 6-month period of conducting tests. The peaks of larval population of the two species were in the 10th and 21st weeks under temperature of 25 ± 2 °C, RH of 60 ± 5% and a photoperiod of 12:12, D:L. The pattern of fluctuation in the parasitism percentage in the larval population of the two moth species host was inconsistent with each other. The parasitism percentage was higher on the larval population of E. kuehniella than larval population of P. interpunctella. In E. kuehniella, the host population change rates were − 0.1 in the 10th and 15th weeks, while the highest rates were 0.1 and 0.8 in the 12th and 3rd weeks. The lowest level of parasitoid balance in the larval population of E. kuehniella and P. interpunctella was recorded in the 11th and 15th weeks, and the highest levels were recorded in the 20th and 24th weeks, respectively. The maximum probability rate of parasitoid non-establishment in E. kuehniella larval population was zero during the entire period and for P. interpunctella until the 22nd week.ConclusionIn fact, there was a strong correlation between establishment success and the severity of host and parasitoid populations. Only in the larval population of P. interpunctella, the maximum probability of failure of establishment was 11.5%, due to the occurrence of the maximum population of parasitized larvae and the minimum ratio of non-parasitic and parasitic larvae.

Effects of parasitism by the braconid wasp, Bracon hebetor (Hymenoptera: Braconidae), on the host hemolymph and phenoloxidase activation of the rice moth, Corcyra cephalonica larvae (Lepidoptera: Pyralidae)

BackgroundBiological control of insect pests is an economic, eco-friendly and harmless approach to integrated pest management strategies. Bracon hebetor (Say) (Hymenoptera: Braconidae) considers a polyphagous ectoparasitoid of various pests of the order Lepidoptera. The parasitized host insects' defense mechanisms are triggered as a result of the parasitic wasps' injury and penetration. Thus, induce the host cellular and humoral immune responses through a blend of secretions injected into the host body. The present study was designed to evaluate the efficacy of B. hebetor on the immune response of the parasitized full grown larval instar of the rice moth, Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) under natural envenomation.ResultsOn cellular level, the ultrastructure examination of the hemocytes displayed a considerable structural deformation in hemocyte morphology of the detected types of the hemocytes. Moreover, the effects of parasitism on both differential hemocyte counts (DHCs) and total hemocyte counts (THCs) were investigated. The number of prohemocytes (PR) (40.33 ± 5.61, 43 ± 8.33 and 26 ± 2.31) was statistically differed after (24, 48 and 72 h) of parasitism, respectively, compared to unparasitized (31.33 ± 6.49) larvae. Similar observations were recorded in plasmatocytes (PL) before and after the parasitism. However, Spherulocytes (SP) and Oenocytoids (OE) were recorded in the hemolymph with little abundance. On the other side, the quantitative analysis of total hemolymph proteins (THP) provoked a significant effect of considering parasitized and non-parasitized larvae. As the Phenoloxidase (PO) cascade plays a critical role in immune defenses, so the substantial activation of PO in the host's hemolymph following successive hours of parasitism compared to unparasitized larvae using L-DOPA as a substrate indicated the induction of larval immune system. The parasitized larvae showed a gradual increase in the PO activity (0.442 ± 0.103) after 24 h reached up to (1.482 ± 0.272) at the end of parasitism (72 h) in comparable to unparasitized larvae (0.177 ± 0.0.044).ConclusionThe present investigations clarified the efficacy of B. hebetor parasitism on the host immune mechanism, which will enable the progress of sustainable stored product protection approaches for the control of an important pest rice moth C. cephalonica.

The Feeding Behaviour of Gall Midge Larvae and Its Implications for Biocontrol of the Giant Reed: Insights from Stable Isotope Analysis.

The gall midge Lasioptera donacis, whose larval stage interferes with the reed's leaf development, is a potential candidate agent for the biological control of Arundo donax. Reed infestation is always associated with the presence of a saprophytic fungus, Arthrinium arundinis, which is believed to provide food for the larvae. Larvae also interact with a parasitic nematode, Tripius gyraloura, which can be considered its natural enemy. To deepen our knowledge of the plant-fungus-insect trophic interactions and to understand the effects of the nematode on midge larval feeding behaviour, we applied stable isotope analysis, one of the most effective methods for investigating animal feeding preferences in various contexts. The results showed that on average the fungus accounted for 65% of the diet of the midge larvae, which however consumed the reed and the fungus in variable proportions depending on reed quality (expressed as the C:N ratio). No differences in feeding behaviour were observed between parasitised and non-parasitised midge larvae, indicating that nematodes have no effect in this regard. Due to its trophic habits, L. donacis could be an effective control agent of A. donax and these results need to be considered when implementing biological control measures.

Irradiation and parasitism affect the ability of larval hemocytes of Anastrepha obliqua for phagocytosis and the production of reactive oxygen species.

The development of the parasitoid Doryctobracon crawfordi (Viereck) (Hymenoptera: Braconidae) in Anastrepha obliqua (McQuart) (Diptera: Tephritidae) larvae is unviable in nature; however, if the host larva is irradiated at 160 Gy, the parasitoid develops and emerges successfully. This suggests that radiation affects the immune responses of A. obliqua larvae, while the underlying mechanisms remain to be revealed. Using optical and electronic microscopies we determined the number and type of hemocyte populations found inside the A. obliqua larvae, either nonirradiated, irradiated at 160 Gy, parasitized by D. crawfordi, or irradiated and parasitized. Based on flow cytometry, the capacity to produce reactive oxygen species (ROS) was determined by the 123-dihydrorhodamine method in those hemocyte cells. Five cell populations were found in the hemolymph of A. obliqua larvae, two of which (granulocytes and plasmatocytes) can phagocytize and produce ROS. A reduction in the number of cells, mainly of the phagocytic type, was observed, as well as the capacity of these cells to produce ROS, when A. obliqua larvae were irradiated. Both radiation and parasitization decreased the ROS production, and when A. obliqua larvae were irradiated followed by parasitization by D. crawfordi, the reduction of the ROS level was even greater. In contrast, a slight increase in the size of these cells was observed in the hemolymph of the parasitized larvae compared to those in nonparasitized larvae. These results suggest that radiation significantly affects the phagocytic cells of A. obliqua and thus permits the development of the parasitoid D. crawfordi.

Behavior of Aedes albifasciatus (Diptera: Culicidae) larvae from eggs with different dormancy times and its relationship with parasitism by Strelkovimermis spiculatus (Nematoda: Mermithidae).

Aedine mosquitoes go through unfavorable periods as dormant eggs. However, extended dormancy times lead to a depletion of egg reserves, which might be partially compensated by changes in larval-feeding behavior. Changes in larval behavior may also be induced by parasitism by mermithids probably as a strategy to reduce the impact of the parasite on the host. The most abundant floodwater mosquito in southern South America is Aedes albifasciatus (Diptera: Culicidae), a species naturally parasitized by Strelkovimermis spiculatus (Nematoda: Mermithidae). This study aimed to evaluate the behavior of fourth-instar larvae of Ae. albifasciatus from eggs with 2, 4, and 6months of dormancy, parasitized or not by S. spiculatus. To achieve this, eight categories of behavior were defined, and then, each individual was observed for 5min, and its behaviors and their duration were recorded. The behaviors with the highest percentage of observation time were still (50.1%) and wriggle swimming (33.6%), while those with the lowest percentage of observation time were those involving active foraging (less than 8%). A great variability in the behaviors was observed among individuals within the same treatment. The type and duration of each behavior did not vary in relation to egg dormancy time or parasitism, but parasitism affected the level of larval activity. Parasitized larvae performed, on average, fewer behaviors than non-parasitized larvae. This result supports the hypothesis that S. spiculatus parasitism affects the behavior of Ae. albifasciatus larvae by reducing their level of activity.

Nesting Efectiveness of Red Mason Bee Osmia rufa (L.) in Reed Stalk Phragmites australis (Cav.)

Abstract The research enabled the optimal parameters of reed stalks Phragmites australis (Cav.) Trin. ex Steud, to be determined and then be used for rearing red mason bees (Osmia rufa L.). During research conducted in 2015 and 2016, 3,293 reed stalks were occupied by red mason bees, and 19,461 nest cells were counted in the stalks, of which there were 16,222 non-parasitized larvae. The mean weight of one cocoon was 0.085 g. The mean length of the reed stalk occupied by the red mason bee was 169.45 mm, its mean diameter was 6.11 mm and the mean number of cells in the reed was 5.47. The mean length of the first cell, the vestibulum, was 38.38 mm. The mean degree of parasitism was 0.16. The length of the reed stalk positively correlated with the number of cells in the reed nest (r=0.543), vestibulum length (r=0.392) and cocoon mass (r=0.362) The diameter of the reed positively correlated with the mass of the cocoon (r=0.536), the number of cells in the reed (r=0.406) and vestibulum length (r=0.254). Correlation between degree of parasitatism and length, and diameter of the reed was insignificant. Reed stalks with a length of 150 to 200 mm and a diameter of 5–7 mm are recommended for the choice of red mason bee nesting material.

Host discrimination in the fruit fly parasitoid Diachasmimorpha longicaudata: evidence from virgin female behaviour and egg distribution patterns.

Many parasitoid species discriminate already parasitized hosts, thus avoiding larval competition. However, females incur in superparasitism under certain circumstances. Superparasitism is commonly observed in the artificial rearing of the parasitoid Diachasmimorpha longicaudata, yet host discrimination has been previously suggested in this species. Here, we addressed host discrimination in virgin D. longicaudata females in a comprehensive way by means of direct and indirect methods, using Ceratitis capitata and Anastrepha fraterculus which are major fruit fly pests in South America. Direct methods relied on the description of the foraging behaviour of females in arenas with parasitized and non-parasitized host larvae. In the indirect methods, healthy larvae were offered to single females and the egg distributions were compared to a random distribution. We found that D. longicaudata was able to recognize parasitized host from both host species, taking 24 h since a first parasitization for A. fraterculus and 48 h for C. capitata. Indirect methods showed females with different behaviours for both host species: complete discrimination, non-random (with superparasitism), and random distributions. A larger percentage of females reared and tested on A. fraterculus incurred in superparasitism, probably associated with higher fecundity. In sum, we found strong evidence of host discrimination in D. longicaudata, detecting behavioural variability associated with the host species, the time since the first parasitization and the fecundity of the females.

Larval parasitic copepods affect early life history traits of a temperate clingfish.

Larval copepods are frequent parasites that infest fish larvae along the Chilean coast. Because these parasites develop on fish during the early development, when their bodies are fragile and in a recent stage, they can affect the fishes' early life history traits (ELHT). The goal of this study was to determine the effect of parasitic copepods on the ELHT of the larvae of the clingfish Gobiesox marmoratus (Teleostei: Gobiesocidae) using otolith microstructure analysis. Ichthyoplankton samples were collected during austral winter (July and August 2012), in the inner shelf waters off Valparaiso Bay, central Chile. A total of 95 non-parasitized larvae (NPL) and 95 parasitized (PL) with copepods were randomly selected for subsequent analyses. Parasitized larvae of G. marmoratus were larger than NPL. The right otolith tended to be larger than the left otolith in the fish larvae, but with a higher asymmetry in PL. The PL showed larger otoliths-at-size than the NPL, particularly in smaller larvae (< 8mm of standard length, SL). Nonetheless, parasitized larvae larger than 8mm SL showed the opposite trend that is smaller-at-size otoliths than NPL. The Gompertz models indicated that the asymptotic length of NPL doubled the length of PL; this suggests that parasitic copepods affect the maximum size attained by the PL. In conclusion, parasitic copepods negatively affect the ELHT of G. marmoratus larvae and the greater asymmetry can be attributed to parasitism.

Parasitism-Mediated Interactions Between the Ring-Legged Earwig and Sugarcane Borer Larvae.

Sugarcane is one of the most economically important crops in Brazil. The damage caused by Diatraea saccharalis (F.) (Lepidoptera: Crambidae) results in high costs for its production. Cotesia flavipes Cameron (Hymenoptera: Braconidae) is the most efficient means of controlling this pest. The prohibition of burning and the preservation of soil cover through non-tillage practices increased many population densities of natural enemies of the sugarcane borer (SCB) in sugarcane fields. Euborellia annulipes (Lucas) (Dermaptera: Anisolabididae) is a predator found in sugarcane fields and is associated with predation of SCB. This study aimed to evaluate the influence of parasitism of D. saccharalis by C. flavipes on predation by E. annulipes adult females. For this purpose, fourth instar sugarcane borer larvae were exposed to parasitism over a 5-day period or were not parasitized. Subsequently, the prey was supplied to the predator in arenas under choice and no-choice conditions and under different densities to evaluate the feeding preference over a 12-h period and functional response of the predator over a 24-h period. E. annulipes killed 2-3 fourth instar larvae of the sugarcane borer over a 12-h period. SCB parasitism did not affect the feeding preference of the predator and did not alter the type II functional response. However, the handling time of the predator was lower (1.943 h) and there was a higher T/Th ratio on the non-parasitized larvae (12.352). Our findings indicate E. annulipes as a promising biological control agent of D. saccharalis, with a potential to assist in the sugarcane borer management approaches and could to reduce the costs of parasitoid release in the field.

Effect of Temperature on the Immature Developmental Time and Adult Longevity of Apanteles Galleriae Wilkinson (Hym. Braconidae)

The greaterwax moth, Galleria mellonella (L.), is a known pest of the Egyptian and worldwide beekeeping. The solitary endoparasitoid, Apanteles galleriae Wilkinson is one of important natural enemies of the pest. The study aimed to determine the effect of temperatures under near-continuous darkness on the immature developmental time, growth of parasitized larvae and adult longevity of A. galleriae. The duration of developmental period of male was less than that of female irrespective of temperatures. At 20°C temperature and 75% RH, the duration ranged from 21.7 days for males and 23.8 days for females, decreased to 18.1 and 19.2 days at 25°C,17.1 and 18.1 days at 27°C and 16.0 and 17.3 days at 30°C, respectively. Parasitism by A. galleriae retards the growth of G. mellonella larvae. The initial weight of wax moth larva at the time of parasitization was 3.5±0.2 mg and was significantly lower (9.9±0.7 mg) prior to parasitoid egression than that of the non-parasitized larva (89.5±5.2 mg). The parasitized host larvae eventually weighed up to 70 to 88% less than the non-parasitized larvae. The adults survived starvation, water- or one -day honey feeding for few days, while they lived for significantly longer periods when they provided daily with pure honey. Longevity of females fed on pure honey was significantly longer than males at all tested temperatures. The results may be of value for developing mass rearing techniques and biological control of wax moths by A. galleriae.

Immune response and susceptibility to Cotesia flavipes parasitizing Diatraea saccharalis larvae exposed to and surviving an LC25 dosage of Bacillus thuringiensis

Biological control using entomopathogens and natural enemies is an ecofriendly method for pest management in agriculture. Biological control agents often can be simultaneously employed and compatibility between agents may improve pest suppression. We investigated the influence of the entomopathogen Bacillus thuringiensis (Bt) on the immune system of the sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) to determine if such changes impact parasitization by Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae). The immune response of surviving D. saccharalis larvae fed with an LC25 dosage of a Bt-based biopesticide (Dipel®) was analyzed (total hemocyte count, hemocyte adhesion, and activities of phenoloxidase and lysozyme). Furthermore, the suitability of surviving Bt-fed larvae as hosts for C. flavipes was assessed by measuring parasitoid attributes such as number and size of teratocytes, weight of pupae, length of adult female tibia and number of emerged adults. Total hemocyte count, but not hemocyte adhesion, total protein content and phenoloxidase activity increased in the hemolymph of non-parasitized Bt-fed larvae (Bt-NP) compared to control larvae (NBt-NP). Lysozyme activity increased only after parasitization without Bt exposure (NBt-P). After parasitization, the immunological parameters activated in Bt-NP larvae decreased to levels at or below those observed in control larvae, showing that C. flavipes can regulate the activated immune response of Bt-fed larvae. The development of C. flavipes was not impaired in Bt-fed larval hosts (Bt-P); no changes were observed for teratocyte size, weight of pupal mass, length of hind tibia and number of adults emerged.

Parasitic copepods affect morphospace and diet of larvae of a temperate reef fish.

The effects of ectoparasites on larvae of the clingfish Gobiesox marmoratus were evaluated at the dietary and morphometric levels. The larvae and ectoparasites were collected by nearshore plankton samplings during October, November and December 2013 off El Quisco Bay, central Chile. The standardized abundance of total larvae and those ectoparasitized larvae (PL) was positively related and high parasite prevalence was found throughout the sampling period (up to 38%). Geometric morphometrics analyses indicate main changes in the shape through early ontogeny and subtle but significant variations between PL and non-parasitized larvae (NPL). Prey composition varied between PL and NPL; small size (<6 mm standard length, LS ) parasitized larval G. marmoratus ate mostly gastropod larvae, whereas small non-parasitized specimens ate mainly cirripede nauplii. All larger (>8 mm LS ), pre-settlement stages parasitized by Trifur and, or Caligus copepods had content in their gut, suggesting that ectoparasites did not diminish prey capture in host with larger size. Morphometric and dietary changes occurring during larval development were decoupled, both for PL and NPL. The maintenance of a slender, more hydrodynamic body through pelagic development and the ingestion of less-mobile prey in PL suggests non-lethal effects of ectoparasitism on rocky-reef fish larvae.

An endoparasitoid wasp influences host DNA methylation

Parasitism by endoparasitoid wasps changes the expression of various host genes, and alters host immune and developmental processes. However, it is not clearly understood how parasitism changes host gene expression in a whole genome scale. This study focused on an epigenetic control of Cotesia plutellae, an endoparasitoid wasp, against its host, Plutella xylostella. Two DNA methyltransferases (DNMT-1 and DNMT-2) are encoded in the genome of P. xylostella. In addition, methyl-binding domain proteins (MBDs) and DNA demethylation factor, ten-eleven translation protein (TET) are encoded. DNA methylation of P. xylostella genomic DNA was confirmed by restriction digestion with Gla I specific to 5-methylcytosine. DNA methylation intensity in parasitized (P) larvae was decreased compared to that in nonparasitized (NP) larvae, especially at late parasitic stage, at which expression levels of both DNMT-1 and DNMT-2 were also decreased. DNA demethylation of P. xylostella was confirmed in both NP and P larvae by restriction digestion with PvuRts1I recognizing 5-hydroxymethyl cytosine. Parasitism also suppressed expression levels of TET and MBDs. Treatment of 5-aza-2′-deoxycytidine (AZA) reduced DNA methylation intensity of NP larvae, causing suppression of hemocyte-spreading behavior and delay of immature development. RNA interference of DNMT-1 or DNMT-2 mimicked the adverse effects of AZA.

Differential responses of Helicoverpa armigera C-type immunlectin genes to the endoparasitoid Campoletis chlorideae.

The C-type lectins mediate nonself recognition in insects. The previous studies focused on host immunlectin response to bacterial infection; however, the molecular basis of immunlectin reactions to endoparasitoids has not been elucidated. The present study investigated the effect of parasitization by Campoletis chlorideae on hemagglutination activity (HA; defined as the ability of lectin to agglutinate erythrocytes or other cells), and transcriptional expression of C-type immunlectin genes in the larval host, Helicoverpa armigera. Parasitization induced four- to eightfold higher HA in the parasitized larvae, compared to nonparasitized larvae at days 2 and 6 postparasitization (PP), however inhibited HA at other days PP. Eight C-type lectins were differentially expressed in different host developmental stages, from feeding to wandering stage. The mRNA levels of HaCTL1, HaCTL3, HaCTL4, and HaCTL5 were upregulated and HaCTL2 and HaCTL7 were downregulated. Tissue analysis showed that HaCTLs were mainly expressed in fat body or hemocytes, while HaCTL5 was highly expressed in testes. The effects of parasitization on the lectin expression patterns differed. Lectins except HaCTL6 or HaCTL5 were significantly down- or upregulated in parasitized larvae at day 4 or 6 PP compared with that of nonparasitized larvae. We infer from our results that C-type immunlectins are involved in host-parasitoid interactions, and parasitization alter host immunlectin levels both in inhibiting and promoting host immune defenses to endoparasitoids. These immunlectin genes indicated an altered physiological status of the host insect, depending on developmental stage, tissue, and parasitization.

A viral histone H4 suppresses insect insulin signal and delays host development

Parasitization by an endoparasitoid wasp, Cotesia plutellae, alters host development of Plutella xylostella by extending larval period and preventing metamorphosis. Insulin signal plays a crucial role in mediating insect development and controlling blood sugar level in insects. In this study, three insulin-like peptide genes (PxILP1-3) were predicted from the genome of P. xylostella. However, only PxILP1 was confirmed to be expressed in P. xylostella. Starvation suppressed the expression level of PxILP1 and up-regulated plasma trehalose level. RNA interference against PxILP1 mimicked starvation effect and extended the larval period of P. xylostella. Parasitized larvae exhibited significantly lower levels of PxILP1 expression compared to nonparasitized larvae. Injection of wasp-symbiotic polydnavirus C. plutellae bracovirus (CpBV) also suppressed PxILP1 expression and extended the larval period. Injection of a viral segment (CpBV-S30) containing a viral histone H4 (CpBV-H4) also suppressed PxILP1 expression. Co-injection of CpBV-S30 and double-stranded RNA (dsCpBV-H4) specific to CpBV-H4 rescued the suppression of PxILP1 expression. Injection of CpBV-S30 significantly extended larval development. Co-injection of CpBV-S30 with dsCpBV-H4 rescued the delay of larval development. Injection of a bovine insulin to parasitized larvae prevented parasitoid development. These results indicate that parasitism of C. plutellae can down-regulate host insulin signaling with the help of parasitic factor CpBV-H4.

Baculoviral p94 homologs encoded in Cotesia plutellae bracovirus suppress both immunity and development of the diamondback moth, Plutellae xylostella.

Polydnaviruses (PDVs) are a group of insect DNA viruses, which exhibit a mutual symbiotic relationship with their specific host wasps. Moreover, most encapsidated genes identified so far in PDVs share homologies with insect-originated genes, but not with virus-originated genes. In the meantime, PDVs associated with 2 wasp genera Cotesia and Glytapanteles encode some genes presumably originated from other viruses. Cotesia plutellae bracovirus (CpBV) encodes 4 genes homologous to baculoviral p94: CpBV-E94k1, CpBV-E94k2, CpBV-E94k3, and CpBV-E94k4. This study was conducted to predict the origin of CpBV-E94ks by comparing their sequences with those of baculoviral orthologs and to determine the physiological functions by their transient expressions in nonparasitized larvae and subsequent specific RNA interference. Our phylogenetic analysis indicated that CpBV-E94ks were clustered with other E94ks originated from different PDVs and shared high similarity with betabaculoviral p94s. These 4 CpBV genes were expressed during most developmental stages of the larvae of Plutella xylostella parasitized by C. plutellae. Expression of these 4 E94ks was mainly detected in hemocytes and fat body. Subsequent functional analysis by in vivo transient expression showed that all 4 viral genes significantly inhibited both host immune and developmental processes. These results suggest that CpBV-E94ks share an origin with betabaculoviral p94s and play parasitic roles in suppressing host immune and developmental processes.

Development ofMicroplitis similis(Hymenoptera: Braconidae) on Two Candidate Host Species,Spodoptera lituraandSpodoptera exigua(Lepidoptera: Noctuidae)

Microplitis similis Lyle (Hymenoptera: Braconidae) is a solitary endoparasitic braconid that generally parasitizes larvae of Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) and many other noctuid species. To understand host preference, fitness, and the effects of M. similis on the hosts, we compared percentage parasitism, development periods, and the effects on host growth in candidate noctuid species. We found high levels of parasitism of S. exigua and Spodoptera litura (F.) larvae but not of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) larvae. The parasitoid wasp larvae took similar amounts of time for development on S. exigua and S. litura larvae, i.e., 13.87 ± 0.15 and 13.69 ± 0.42 d, respectively. Compared with the control larvae, the growth and development of the hosts were severely affected. The hosts were able to molt to 4th instars after being parasitized as early 3rd instars, but were unable to develop to the 5th instar. The body weight was similar between parasitized and non-parasitized larvae within the first 4 d (3 d in S. litura) but later began to show a significant difference from the 5th day on (4th day in S. litura). The host larvae eventually weighed up to 50 to 80% less than the non-parasitized larvae. Furthermore, the host larvae lived for an extended period in the same instar after egression of the parasitoid, but the body mass did not increase.

X-ray doses to safely release the parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae) reared on Anastrepha fraterculus larvae (Diptera: Tephritidae)

Diachasmimorpha longicaudata is a koinobiont larval parasitoid that is currently used to control fruit flies of the genera Anastrepha, Ceratitis and Bactrocera. In the rearing process, a fraction of the host larvae that are exposed to parasitoids escape from parasitism and develop into viable and fertile flies. This creates the need to eliminate emerging flies before the parasitoids are shipped for release, increasing costs due to additional handling steps. Exposure of fly eggs or larvae to gamma-irradiation before they are parasitised has been used to reproductively sterilise hosts, or even inhibit their emergence. Our aim was to determine whether X-ray radiation applied to Anastrepha fraterculus third instar larvae before they are exposed to parasitoids, inhibits fly emergence in non-parasitised larvae without affecting the performance of the parasitoids that emerge from parasitised larvae. Three X-ray doses: 6250.2 R, 8333.6 R and 10417 R (equivalent to 60, 80 and 100 Gy, respectively) and one γ-ray dose (100 Gy) were tested. Fly emergence decreased with increasing doses of radiation, showing null values for the higher X-ray dose and the dose of 100 Gy. Irradiation showed either no impact or a positive effect on parasitism rate and fecundity. Sex rate was biased towards females in almost every dose. We conclude that the two types of radiation evaluated here were equally effective in suppressing fly emergence with no detrimental effects on the biological quality of the produced parasitoids. X-rays offer an alternative method of irradiation than the conventional radiation source, i.e. γ-rays. These results represent a significant improvement in the development of a biological control programme against A. fraterculus.

THE ENDOPARASITOID Campoletis chlorideae INDUCES A HEMOLYTIC FACTOR IN THE HERBIVOROUS INSECT Helicoverpa armigera

Although lysis of invading organisms is a major innate form of immunity used by invertebrates, it remains unclear whether herbivorous insects have hemolysin or not. To address this general question, we tested the hemolytic (HL) activity of the hemolymph and tissue extracts from various stages of the polyphagous insect Helicoverpa armigera (Hübner) against the erythrocytes from chicken, duck, and rabbit. An HL activity was identified in the hemolymph of H. armigera larvae. Further studies demonstrated that the HL activity is proteinaceous as it was precipitable by deproteinizing agents. Hemolysins were found in Helicoverpa egg, larva, pupa, and adult, but the activity was higher in feeding larvae than in molting or newly molted larvae. Hemolysins were distributed among a variety of larval tissues including salivary gland, fat body, epidermis, midgut, or testes, but the highest activity was found in salivary gland and fat body. Relative to nonparasitized larvae, parasitization of H. armigera larvae by the endoparasitoid Campoletis chlorideae Uchida induced a 3.4-fold increase in the HL activity in the plasma of parasitized host at day two postparasitization. The present study shows the presence of a parasitoid inducible HL factor in the parasitized insect. The HL activity increased significantly in H. armigera larvae at 12 and 24 h postinjection with Escherichia coli. We infer the HL factor(s) is inducible or due to de novo synthesis, which means that the HL factor(s) is associated with insect immune response by inhibiting or clearance of invading organisms.