Pauline O. Lawrence

Host vibration — A cue to host location by the parasite, Biosteres longicaudatus

SummaryBiosteres longicaudatus Ashmead (Hymenoptera: Bracon dae) is a solitary endoparasite of Anastrepha suspensa larvae (Diptera: Tephritidae), which live in fruit tissue. Larvae make andible noises within macerated fruit or larval medium in which they are reared. Parasite females readily located normal, mobile larvae and spent a mean of 16.5±4.7 min/visit to parasitize these hosts. In contrast, females were unable to locate etherized or dead hosts and abandoned them after only 1.9±0.9 and 2.3±0.8 min, respectively. Females of all ages, with and without oviposition experience, exhibited non-random search and ovipositor probe behaviors in response to artifically created vibration. This response was influenced primarily by the number of mature eggs in the ovaries. These findings suggest that 1) an accumulation of mature eggs in the ovaries increase the appetitive drive of females to find and oviposit in hosts and 2) host sound/vibration produced either by movement of hosts through the medium and/or by the rasping mouth hooks during feeding. is used by parasites as a releaser for host finding behavior as well as a cue to the location of the host within the substrate.

HORMONAL EFFECTS ON INSECTS AND OTHER ENDOPARASITES IN VITRO

SummaryMetamorphic and reproductive events in vertebrates and invertebrates are under endocrine control and are often correlated with developmental, behavioral, or reproductive changes in the parasites living in or on these hosts. This paper reviews selected examples ofa) host hormone mediated influences on endoparasites in vivo,b) host hormone effects in vitro on protozoan, helminth, and insect endoparasites, andc) identifies possible relationships in hormone effects across parasite taxa. The significance of studies on endoparasites in vitro in relation to the impact of host hormones, antihelminthic, and prophylactic drugs on parasite growth and proliferation will also be addressed. A review of the literature indicates only limited studies have been done in vitro in an attempt to elucidate the bases of reported host hormone influences on endoparasites in vivo. Steroid hormones of hosts seem to stimulate growth, molting or encystment or both of helminth, insect, and protozoan parasites. Vertebrate steroids such as estrogen, testosterone, and progesterone had primarily reproduction- or growth-promoting effects or both on protozoan and nematode parasites. Insect ecdysteroids such as ecdysone, 20-hydroxyecdysone, and makisterone were the most widely studied steroids in vitro and induced growth or molting or both of cestode, nematode, and insect parasite larvae. Although juvenile hormone (JH III) stimulated growth in the protozoan and nematode parasites tested, the analogue methoprene and JH precursors, farnesal, farnesol, and farnesol methyl ether had various effects. Biogenic amines also varied in their effects on the nematode parasites tested, while the peptide hormone, insulin, stimulated growth in the protozoans tested. The evidence for in vitro effects of host hormones on their natural endoparasites is patchy at best. Additional studies are needed to identify the biochemical bases for the numerous host hormone mediated effects on parasites.

Biosteres longicaudatus: Developmental dependence on host (Anastrepha suspensa) physiology

Abstract Larvae of Anastrepha suspensa that were in the first day of the third instar were parasitized by females of the solitary endoparasitoid, Biosteres longicaudatus. At the end of the 6-hr oviposition period, larvae were ligated posterior to the ring gland so that some larvae had parasitoids anterior to the ligature while in others, the parasitoids were in the abdomen, posterior to the ligature. Ninety-two percent of the parasitoids anterior to the ligature hatched to the first through third instars. Parasitoids posterior to the ligature had a 75% egg hatch to the first instar only. No larval molts to the second or subsequent instars occurred in these parasitoids. Upon parabiosis to 3-day-old, unparasitized host pupae, the ligated larvae pupated and 97% of the first-instar parasitoids in these parabiosed larval abdomens molted to the second instar. Newly laid parasitoid eggs transplanted to 3-day-old pupal hosts had less than one-third of the egg hatch of those transplanted to first-day third-instar hosts. The data implicate the physiological state of the host (vis-a-vis pupation and associated events) as being an important factor in the development of the endoparasitoid.

Intraspecific competition among first instars of the parasitic wasp Biosteres longicaudatus

SummaryFemales of the solitary endoparasitic wasp Biosteres longicaudatus sometimes deposit >1 egg (superparasitism) in each larva of the Caribbean fruit fly host, Anastrepha suspensa. As host density increases, there is an inverse relationship between the level of superparasitism and the number of progeny produced/female. Larval parasitoid competition in superparasitized hosts causes an abrupt decline from >1 to 1 or <1 parasitoid/host 24–36 h before the surviving parasitoid larva molts to the 2nd instar. The mechanism by which supernumaries are eliminated was investigated by indirect, in vivo and direct, in vitro methods. There is no apparent competition between parasitoid eggs of the same age. Parasitoid first instars utilize their heavily sclerotized mandibles to eliminate competitors, some of which are subsequently encapsulated by the host. First instars in vitro produce a substance that kills conspecifics. Presumably, this substance is secreted into the surrounding medium. One of each pair of parasitoid first instars, evenly matched for age and size, may live up to 6.4 days longer and grow to 0.13 mm larger than the other. Thus, B. longicaudatus, like other solitary endoparasitoids eliminates competitors by both combat and interference competitions. The latter case, presumably involves allelochemical toxins against conspecifics in the absence of physical encounter.

Ecdysteroid titres and integument changes in superparasitized puparia of Anastrepha suspensa (Diptera: Tephritidae)

Larval-pupal apolysis of Anastrepha suspensa is inhibited when superparasitized (up to 7 parasites/host) by the solitary endoparasite Biosteres longicaudatus. Ecdysone 20-monooxygenase activity was similar in superparasitized hosts and controls and could not explain the significantly (P < 0.05) high ecdysteroid titres, especially in 36–48-h-old hosts. In the control, synthesis of the outer and inner epicuticules coincided with a significantly (P < 0.01) low ecdysteroid titre. Presumably, an inhibitory effect of high ecdysteroid titres and the abnormal proliferation of microvilli at the cuticle-epidermis interface led to the inability of superparasitized hosts to synthesize new cuticle. Moreover, the migration and exocytosis of vesicles observed at the cell apices in the control did not occur in superparasitized individuals which did not apolyse. Viral particles, presumably of the rhabdovirus type (300 × 80 nM) proliferated in large vesicles bordering the basement membrane of epidermal cells in superparasitized puparia. These viruses may have affected the migration of vesicles to the cell apices and the activation of the moulting fluid. Pox virus-like particles (240 nM in diameter) were also observed in haemocytes adjacent to the epidermal cells. Superparasitized A. suspensa were unable to initiate larval-pupal apolysis due presumably, to: (a) their inability to secrete moulting gel and (b) high ecdysteroid titres that may also have inhibited the synthesis of a new cuticle.

Comparative analysis of selected genes from Diachasmimorpha longicaudata entomopoxvirus and other poxviruses

Abstract The Diachasmimorpha longicaudata entomopoxvirus (DlEPV) is the first symbiotic EPV described from a parasitic wasp. The DlEPV is introduced into the tephritid fruit fly larval host along with the wasp egg at oviposition. We sequenced a shotgun genomic library of the DlEPV DNA and analyzed and compared the predicted protein sequences of eight ORFs with those of selected poxviruses and other organisms. BlastP searches showed that five of these are homologous to poxvirus putative proteins such as metalloprotease, a putative membrane protein, late transcription factor-3, virion surface protein, and poly (A) polymerase (PAP) regulatory small subunit. Three of these are similar to those of other organisms such as the gamma-glutamyltransferase (GGT) of Arabidopsis thaliana, eukaryotic initiation factor 4A (eIF4A) of Caenorhabditis briggsae and lambda phage integrase ( λ -Int) of Enterococcus faecium. Transcription motifs for early (TGA,A/T,XXXXA) or late (TAAATG, TAAT, or TAAAT) gene expression conserved in poxviruses were identified with those ORFs. Phylogenetic analysis of multiple alignments of five ORFs and 20 poxvirus homologous sequences and of a concatenate of multiple alignments suggested that DlEPV probably diverged from the ancestral node between the fowlpox virus and the genus B, lepidopteran and orthopteran EPVs, to which Amsacta moorei and Melanoplus sanguinipes EPV, respectively, belong. The DlEPV putative GGT, eIF4A, and λ -Int contained many conserved domains that typified these proteins. These homologues may be involved in either viral pathogenicity or enhancing parasitism via the gamma-glutamyl cycle and compensation of eIF4A levels in the parasitized fly, or via the integration of a portion of the viral genome into the wasp and/or parasitized fly.

Developmental and reproductive biologies of the parasitic wasp, Biosteres longicaudatus, reared on hosts treated with a chitin synthesis inhibitor

Larvae of the Caribbean fruit fly, Anastrepha suspenso Loew (Diptera: Tephritidae) parasitized by the wasp, Biosteres longicaudatus Ashmead (Hymenoptera: Braconidae), were topically treated with diflubenzuron (Dimilin®), a chitin synthesis inhibitor, at 100, 500 and 1000 ppm. Each dosage was initiated during the parasite’s egg stage, each of four larval stages and the pupal stage (pharate pupa + pharate adult). Adults emerging from all immature stages treated with the two higher dosages were significantly (P < 0.05) fewer than the controls. Developmental stages, in decreasing order of susceptibility to diflubenzuron were: egg, first instar, pharate adult, pharate pupa and larval stages two to four. Abnormally developed ovipositors occurred in some adults which emerged from eggs, pharate pupa and pharate adults that were treated with the two higher concentrations. Such females were unable to successfully locate and parasitize hosts. Females with normal ovipositors which emerged from treated eggs and pharate adults were able to find and attack hosts but had significantly fewer eggs in their ovaries and oviposited less than the controls. The F1 eggs laid by these females developed normally and survived to adulthood with no apparent abnormalities.

A 24 kDa parasitism-specific protein from the Caribbean fruit fly, Anastrepha suspensa: cDNA and deduced amino acid sequence.

A 24 kDa parasitism-specific protein (PSP24) was previously reported from the hemolymph of the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae) after parasitization by the wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae). This study was designed to sequence the open reading frame of PSP24 and to determine whether it is encoded by the wasp, fruit fly host or by the entomopoxvirus D1EPV which is normally injected into the host with the wasps egg. Utilizing an existing partial amino acid sequence of PSP24, we obtained two cDNAs by reverse transcription-polymerase chain reaction, from the host hemolymph 48 h post parasitization. The smaller cDNA has an open reading frame (ORF) that encodes 85 amino acids (aa) with a molecular mass of 9711.33 Da and the larger encodes 203 aa with a molecular mass of 23,076 Da. Both cDNAs share a common N-terminus with a signal peptide predictive of secreted proteins, a characteristic that agrees with the observed nature of PSP24. The mature proteins have 39 and 157 aa with deduced molecular masses of 4286.86 Da and 17,651 Da, respectively. Western blots of host hemolymph probed with the anti-PSP24 serum reveal proteins of 0.10 and 0.24 kD, respectively. The discrepancy between the deduced and the observed molecular masses may be explained by their predicted O-linked glycosylation. The amino acid sequences are not homologous with any protein in the available databases. Southern blot hybridization experiments revealed that the proteins are encoded by both the host and the parasite. Furthermore, injection of D1EPV into healthy fruit fly puparia induces the two proteins. Thus, in surprising contrast to an earlier hypothesis that D1EPV encodes PSP24, these results clearly demonstrate that the PSP24 proteins are encoded by wasp and fruit fly but not D1EPV genes. However, their expression is D1EPV induced.

Ecdysteroid levels and integument changes in post-embryonic stages of Anastrepha suspensa

Ecdysteroid levels in larvae and pupae of Anastrepha suspensa (Diptera: Tephritidae) were measured by radioimmunoassay. These levels were correlated with histological changes throughout the development of the post-embryonic stages. Ecdysteroid levels increase rapidly throughout the last-larval instar and on the last day of this stage are 283 pg equivalents of 20-hydroxyecdysone per insect (14.5 ng/g) when wandering behaviour is initiated. At the end of this period the puparium is formed and within 24 h, the ecdysteroid rises to its highest peak (625 pg equivalents of 20-hydroxyecdysone/insect). Larval-pupal apolysis is initiated within 24 h later and the pupal cuticle is then secreted. Two days later, the ecdysteroids reach their lowest levels (75 pg equivalents of 20-hydroxyecdysone/insect or 0.6 ng/g) and most of the larval fat body and other tissues have been histolysed. In 5- to 10-day old pupae ecdysteroid levels again increase and remain relatively high throughout. During this period the larval epidermis is replaced by imaginal epidermis, imaginal discs begin to proliferate and the adult cuticle is secreted. Ecdysteroid levels finally fall 2 days prior to adult emergence. HPLC determinations indicate that 20-hydroxyecdysone is the predominant free ecdysteroid, and along with ecdysone, is readily detectable in all postembryonic stages of this species. An unusually high and unexplained peak of 20-hydroxyecdysone occurs in the pharate adult. This peak probably consists of ecdysone metabolites with retentions similar to that of 20-hydroxyecdysone and to which the antiserum is sensitive.

Age-specific fecundity and offspring survivorship in the Caribbean fruit fly, Anastrepha suspensa , after diflubenzuron treatment

Late third stage (7-day-old) larvae and 1-day-old pupae of Anastrepha suspensa Loew (Dip-tera: Tephritidae) were dipped for 5 min in suspensions of 25% WP Dimilin® (diflubenzuron) at six dosages ranging from 0.003-0.1% a.i. Diflubenzuron caused significant (P < 0.05) mortality during the pupal stages of both groups treated. Consequently, the resulting adult populations were drastically reduced. The incidence of crumpled wings, deformed abdomens and ovipositors in these adults was 2–7 times and 4–9 times higher than the respective controls of treated larvae and pupae. The morphological abnormalities altered mating competitiveness of males and successful copulation and oviposition by females. Fecundity of females was reduced and diflubenzuron effects persisted into the first generation and caused decreased egg viability.Adults fed a 0.1% a.i. diflubenzuron-treated diet for 27 days had no significant mortality, but had decreased fecundity. However, their pattern of oviposition did not differ from the control. Fecundity levels similar to the control’s were achieved 6 days after the treated diet was replaced by normal diet. Diflubenzuron treatment of adults also drastically decreased the survivorship of first-generation individuals between egg-hatch and pupation. However, almost all individuals that pupated emerged as adults. No abnormalities were observed in first-generation adults or in the survivorship of their offspring.