Analysis of mitochondrial COI sequences of the Diachasmimorpha longicaudata (Hymenoptera: Braconidae) species complex in Thailand

Diachasmimorpha longicaudata is an Opiinae parasitoid used to control tephritid fruit flies, which cause tremendous economic losses of fruits worldwide. In Thailand, D. longicaudata is classified as three sibling species, DLA, DLB and DLBB, based on the morphological and biological species concepts but their genetic variation has not been studied. Therefore, we investigated the genetic differentiation of the mitochondrial COI gene to clarify the ambiguous taxonomy of this species complex. The 603‐bp COI region was sequenced from laboratory‐bred colonies and field‐collected specimens from seven locations representing five geographical regions in Thailand. DLA was associated with the host Bactrocera correcta while DLB and DLBB were associated with Bactrocera dorsalis. The interspecific nucleotide differences of COI sequences among the three groups ranged from 6.70% to 7.62% (Kimura 2‐parameter distance), which adequately separates species complexes within the order Hymenoptera and supports the current sibling species classification. The neighbor joining, maximum likelihood and consensus Bayesian phylogenetic trees constructed from COI sequences revealed that the three sibling species of laboratory and field‐collected D. longicaudata are monophyletic with 100% support. The high genetic variation and molecular phylogeny of the COI sequences were shown to discriminate between the D. longicaudata species examined in this study.     

Protein polymorphism in natural populations of Diachasmimorpha longicaudata (Hymenoptera: Braconidae) in Thailand

Diachasmimorpha longicaudata (Ashmead) is a larval parasitoid of tephritid flies and is widely used as a classical biological control agent. We have used allozyme electrophoresis to evaluate the genetic relationships of six populations of D. longicaudata in Thailand. Twelve loci were examined of which 11 were polymorphic in at least some populations, especially that of Nakornratchasima. We observed a complete lack of heterozygotes for seven of the 10 polymorphic loci in the Nakornratchisima female population, and a significant deficiency of heterozygotes at a further two loci. We discuss possible hypotheses for these findings in light of the haplo-diploid sex determination system of these wasps. This revised version was published online in July 2006 with corrections to the Cover Date.     

前裂长管茧蜂个体发育研究

前裂长管茧蜂Diachasmimorpha longicaudata（Ashmead）是许多双翅目实蝇类害虫的重要寄生性天敌。在实验室条件下,以桔小实蝇Bactrocera dorsalis（Hendel）为繁育寄主,系统观测了前裂长管茧蜂个体发育过程中各虫态的特征及其相应的发育历期。观察结果表明,前裂长管茧蜂整个生活史由卵、幼虫、蛹及成虫四个阶段组成。其幼虫可划分为四个龄期,一龄幼虫具几质丁的坚硬头壳和镰刀状的颚,形态特征与行为习性与二、三、四龄幼虫完全不同。前裂长管茧蜂约需20天完成世代发育。卵、一龄幼虫、二龄幼虫、三龄幼虫及四龄幼虫的历期依次为1．91、2．02、1．75、2．04和2．31d;蛹的历期介于8～10d,雌性个体发育时间长于雄性。前裂长管茧蜂通常比未被寄生的寄主迟3～5d羽化,雌、雄蜂在羽化后即能交配、产卵,被寄生的桔小实蝇幼虫体表上会留下明显的深褐色产卵孔,寄主化蛹后产卵孔仍保留在其蛹壳上。     

Spermatogenesis in the parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae)

The study of spermatogenesis in Hymenoptera is limited to few taxa due to limitations to access the material in the right stage for analysis. The information available up to now states that the gametogenesis in males involves a modified meiosis, similar to mitosis. This is due to the haplodiploid sex determination system present in the order, where females are diploid and males are haploid. In the present work, we examined the spermatogenesis process in the parasitoid Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae). We performed squash preparations from testes of individuals from the third larval instar until the adult stage. Some cytological traits, such as the presence of a monopolar spindle, were analysed in detail by means of histological semi‐thin sections. The spermatogenesis in this species comprises an abortive first division with the formation of an anucleated cytoplasmic bud and a nucleated cell, and an equal second division of the nucleated cell, leading to the generation of two mature spermatozoa. The results presented herein provide more information about the process of spermatogenesis in Hymenoptera, which has not been studied in depth in parasitic species.     

Dispersion patterns of Diachasmimorpha longicaudata (Hymenoptera: Braconidae) in citrus orchards in southeast Brazil

We studied the dispersion patterns of the exotic endoparasitoid, Diachasmimorpha longicaudata (Ahsmed) (Hymenoptera: Braconidae), in 1999 (summer) and in 2000 (winter) in a citrus orchard in southeast Brazil. Different population densities of D. longicaudata were released in the centre of the orchard, and their dispersion was determined by using yellow, sticky, rectangular traps, placed in various distances and heights around the release point. Our results suggest that during summer, climatic conditions did not affect dispersion. However, in winter, dispersion rates were positively affected by temperature, and negatively by rainfall. Both estimated dispersal distance and surface were higher in summer than in winter for all release densities. Dispersion peaked at 2000 parasitoids ha^?1 in summer and 8000 parasitoids ha^?1 in winter. The importance of our results for the biological control of fruit flies by augmented or innoculative releases of D. longicaudata in southeast Brazil is discussed.     

Parasitism on medfly by Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) in different guava cultivars

The parasitism efficiency of the Braconidae wasp, Diachasmimorpha longicaudata (Ashmead), was checked on four guava cultivars (Paluma, Sassaoca, Pedro Sato and Kumagai) infested with larvae of medfly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Five blocks of eight fruits, each with two fruits of each cultivar, were put inside C. capitata adult cages, during 2h for oviposition, and a week later, when the larvae inside guavas were developed, the fruits were exposed to parasitoids for 24h. The mean fruit weight, larvae mortality, number of pupae, percentage of medfly and parasitoid emergence were evaluated. There was not statistical difference among cultivars to weight, larvae mortality, number of pupae e emergence of medfly. The percentage of parasitism was higher in Pedro Sato cultivar (19.8%) compared with Kumagai cultivar (2.9%), but it was statistically similar to the other cultivars.     

Mutual interference in the mass-reared fruit fly parasitoid,Diachasmimorpha longicaudata (Hymenoptera: Braconidae)

Diachasmimorpha longicaudata is a solitary endoparasitoid produced in Mexico for the biological control of Anastrepha fruit flies (Diptera: Tephritidae). We studied the effect of mutual interference among conspecific foraging females to better understand the parasitoid-host dynamics established in the mass-rearing system of this species. We used a constant host availability of 60 third instar larvae of Anastrepha ludens (per oviposition unit type Petri dish) that were individually exposed for 3 h to 1, 2, 4, 8, 10, 14, and 20 D. longicaudata females, seven days old without oviposition experience. The following parameters were evaluated: total number of attacked hosts (i.e., larvae with at least one oviposition scar), number of attacked hosts per female, adult emergence percentage, and female proportion. We also performed a second assay at mass-rearing level, contrasting the current proportion used in mass-rearing (~?two host larvae per female) with the alternative treatments (5 and 7.5 host larvae per female). Results showed that the density of females foraging on a patch exhibits an inverse relationship to the number of hosts attacked by one female. The highest values for adult emergence (73.4%), patch exploitation (94.6%), and female proportion (0.86) were obtained in the treatments with 7.5, 6, and 3 host larvae per female, respectively. Under mass-rearing conditions, we found that a lower density of females per cage (~?five larvae per female) reduced superparasitism levels without affecting adult emergence and the proportion of females. This suggests that the mass-rearing efficiency of D. longicaudata could be improved by reducing the density of foraging females.

     

Flight Tunnel Responses of Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) to Olfactory and Visual Stimuli

Cohorts of mass-reared, naive Diachasmimorpha longicaudata, parasitoids of tephritid fruit flies, were released in a laminar airflow wind tunnel to study their responses to visual and olfactory stimuli associated with their host habitat. Parasitoids were five times more likely to land on yellow plastic spheres emitting the odor of ripe, guava fruit (Psidium guajava L.) than to spheres emitting clean air. The rate of landing was not modified by the presence of green artificial leaves adjacent to the spheres in the tunnel or by the inclusion of green leaf volatiles emanating with the guava odors. However, hovering activity was twice as frequent around spheres adjacent to artificial leaves than around isolated spheres. Oviposition activity on spheres emitting guava odor was not affected by the presence of artificial leaves nor by green leaf volatiles. This suggests that inexperienced D. longicaudata may be instinctively attracted to foliage and to fruit odor but that landing (arrestment) and oviposition are influenced more by odor than by the appearance of fruit or foliage. D. longicaudata are not instinctively attracted to larvae of Bactrocera dorsalis in the absence of host-habitat stimuli. More wasp activity occurred around oviposition units containing larvae if the odor of ripe/overripe guava was present. Successful wasp reproduction occurred only in units with guava odor.     

桔小实蝇及其寄生性天敌——前裂长管茧蜂饲养方法

系统介绍了桔小实蝇Bactrocera dorsalis（Hendel）室内饲养的设备、步骤、技术要点、人工饲料配方及环境要求,并以桔小实蝇为寄主,实现了桔小实蝇幼虫一蛹期寄生性天敌前裂长管茧蜂Diachasmimorpha longicaudata（Ashmead）的室内大量繁殖,为进一步利用该蜂防治桔小实蝇打下了良好的基础。     

First record of Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) attacking Toxotrypana curvicauda Gerstaecker (Diptera: Tephritidae)

A new host record is reported for the braconid wasp Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), parasitizing papaya fruit fly larvae Toxotrypana curvicauda Gerstaecker (Diptera: Tephritidae) in México.     

Oviposition Behavior and Conspecific Host Discrimination in Diachasmimorpha longicaudata (Hymenoptera: Braconidae), a Fruit Fly Parasitoid

We studied conspecific host discrimination and oviposition behavior of Diaschasmimorpha longicaudata (Ashmead), in third instar Anastrepha ludens (Loew) under laboratory conditions. The complete process of oviposition in D. longicaudata required an average of 29±11.7 s (Mean±SE), during which time the female remained completely immobile. This contrasts with attempts to oviposit for lesser durations (2-4 s), during which the female constantly moved her antennae and abdomen. In order to determine the host discrimination ability of this species (i.e., the capacity to distinguish between parasitized and non-parasitized hosts), third-instar A. ludens non-parasitized or parasitized 24 h earlier, were exposed simultaneously to individual female wasps with or without previous oviposition experience. An identical test was performed using larvae parasitized 48 h earlier. Both types of females showed a similar behavioral pattern of oviposition, but with significant differences in the number of eggs laid in parasitized and non-parasitized larvae. Experienced females showed reduced incidence of oviposition in parasitized larvae as well as a greater number of probes that did not lead to parasitism (rejection), although this difference was only significant in the 24-h test. This suggests that the host discrimination capacity of this parasitoid is innate and that previous oviposition experience increases the discrimination ability of females.     

Effect of hypoxia and its repercussions in packing pupae of the parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae) for shipment

Hypoxia periods of 6, 12, 24, 48, and 72?h combined with temperatures of 15°C, 20°C and 25°C were applied to Diachasmimorpha longicaudata pupae in packaging conditions. Alternating temperatures of 26–20–26°C were applied to pupae in three sequential 4-h periods less than 12?h of hypoxia, and the effect of hypoxia on the pupae was evaluated under ambient conditions in commercial shipping. The emergence, longevity, fecundity and flight ability of adult parasitoids were recorded. The duration of hypoxia (>24?h) and the higher temperature (25°C) significantly reduced the emergence and longevity of adults. The different temperatures, including the variation in temperature combined with hypoxia, showed no significant effect on fecundity or flight ability. The temperature and humidity recorded during commercial shipping under hypoxia did not show any effect on the emergence of parasitoids. As a consequence, hypoxia was only notably significant after 24?h, which occasionally occurs in practical packaging processes. The use of hypoxia for shipping D. longicaudata pupae can be widely recommended, but long shipping periods and high temperatures should still be avoided.     

Diachasmimorpha longicaudata and D. kraussii (Hymenoptera: Braconidae), potential parasitoids of the olive fruit fly

The olive fruit fly, Bactrocera oleae (Tephritidae), is a significant threat to California's olive industry. As part of a classical biological control program started in 2002, the parasitoids Diachasmimorpha kraussii and D. longicaudata (Hymenoptera: Braconidae) were imported to California from laboratory colonies in Hawaii. Studies on their biology and behavior as parasitoids of the olive fruit fly were conducted in quarantine. Both species tend to oviposit into 2nd and young 3rd instars, with the offspring completing development in the flies’ puparia. Most eggs are deposited in the first two weeks of adult life. Observed lifetime fecundity was low, possibly as a consequence of the relatively poor quality of the harvested olives used as a host substrate. Both pre-imaginal development and adult longevity were limited at constant temperatures above 30°C, which may indicate that these species will have difficulty establishing in the warmest regions of California.     

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.     

Pupal development of Ceratitis capitata (Diptera: Tephritidae) and Diachasmimorpha longicaudata (Hymenoptera: Braconidae) at different moisture values in four soil types

This study aimed to evaluate adult emergence and duration of the pupal stage of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), and emergence of the fruit fly parasitoid, Diachasmimorpha longicaudata (Ashmead), under different moisture conditions in four soil types, using soil water matric potential. Pupal stage duration in C. capitata was influenced differently for males and females. In females, only soil type affected pupal stage duration, which was longer in a clay soil. In males, pupal stage duration was individually influenced by moisture and soil type, with a reduction in pupal stage duration in a heavy clay soil and in a sandy clay, with longer duration in the clay soil. As matric potential decreased, duration of the pupal stage of C. capitata males increased, regardless of soil type. C. capitata emergence was affected by moisture, regardless of soil type, and was higher in drier soils. The emergence of D. longicaudata adults was individually influenced by soil type and moisture factors, and the number of emerged D. longicaudata adults was three times higher in sandy loam and lower in a heavy clay soil. Always, the number of emerged adults was higher at higher moisture conditions. C. capitata and D. longicaudata pupal development was affected by moisture and soil type, which may facilitate pest sampling and allow release areas for the parasitoid to be defined under field conditions.     

Discrimination by Coptera haywardi (Hymenoptera: Diapriidae) of hosts previously attacked by conspecifics or by the larval parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae)

Coptera haywardi (Oglobin) is an endoparasitoid of fruit fly pupae that could find itself in competition with other parasitoids, both con- and heterospecific, already resident inside hosts. In choice bioassays, ovipositing C. haywardi females strongly discriminated against conspecifically parasitised Anastrepha ludens (Loew) pupal hosts. They also avoided pupae previously attacked by Diachasmimorpha longicaudata (Ashmead), a larval–prepupal koinobiont endoparasitoid, and the degree of larval-parasitoid superparasitism had no effect on this avoidance. There was no difference in the number of ovipositor insertions when hosts previously parasitised by a conspecific and D. longicaudata were exposed simultaneously. As females aged the degree of host discrimination declined. An ability to discriminate against pupae previously attacked as larvae suggests low levels of both conspecific and heterospecific competition in the field.     

Interactions Between Augmentatively Released Diachasmimorpha longicaudata (Hymenoptera: Braconidae) and a Complex of Opiine Parasitoids in a Commercial Guava Orchard

A study was conducted to determine the eVect of augmentatively releasing mass-reared Diachasmimorpha longicaudata (Ashmead) on a complex of four co-existing parasitoid species which attack the oriental fruit fly, Bactrocera dorsalis (Hendel). The species belonging to this complex are the egg-pupal parasitoid, Biosteres arisanus (Sonan) and the larval-pupal parasitoids, Biosteres vandenboschi (Fullaway), D. longicaudata and Psyttalia incisi (Silvestri). The study site was a 160-ha commercial orchard of common guava, Psidium guajava L. (cv. Beaumont) located on Kauai island. One year before the release of D. longicaudata, B. arisanus was the dominant parasitoid, accounting for 91.1% of the parasitoids recovered. Despite releases of large numbers of D. longicaudata (600000-800 000 parasitized puparia/ week), B. arisanus continued to account for 90% of all parasitoids recovered from the oriental fruit fly. The larval parasitoid P. incisi may have been reduced as a result of D. longicaudata releases. D. longicaudata was significantly more abundant in over-ripe and rotting fruits compared with freshly fallen fruits. However, no increase in the overall percentage parasitism was observed in any fruit ripeness category. B. arisanus was eY cient at colonizing hosts when fruit fly densities and fruit abundance were relatively low in the orchard. The implications for future augmentative release programmes with D. longicaudata are discussed.