Detection of Pfiesteria spp. by PCR in surface sediments collected from Chesapeake Bay tributaries (Maryland)

In 1997 blooms of Pfiesteria piscicida occurred in association with fish kills and human health problems in tributaries of the Chesapeake Bay (Maryland) and the scientific and media response resulted in large economic losses in seafood sales and tourism. These events prompted the Maryland Department of Natural Resources (MDNR) to begin monitoring for Pfiesteria spp. in water column samples. Real-time PCR assays targeted to the 18S rRNA gene were developed by our laboratories and utilized in conjunction with traditional microscopy and fish kill bioassays for detection of these organisms in estuarine water samples. This monitoring strategy aided in determining temporal and spatial distribution of motile forms of Pfiesteria spp. (i.e. zoospores), but did not assess resting stages of the dinoflagellates’ life cycle. To address this area, a 3-year study was designed using real-time PCR assays for analysis of surface sediment samples collected from several Chesapeake Bay tributaries. These samples were tested with the real-time PCR assays previously developed by our laboratories. The data reported herein suggest a strong positive association between presence of Pfiesteria spp. in the sediment and water column, based on long-term water column monitoring data. P. piscicida is detected more commonly in Maryland's estuarine waters than Pfiesteria shumwayae and sediment ‘cyst beds’ may exist for these organisms.     

Associations between fish health and Pfiesteria spp. in Chesapeake Bay and mid-Atlantic estuaries

In response to concerns that there may be an association between harmful algal bloom (HAB) species and fish health, including the widespread use of fish health as one indicator of a possible HAB warranting further investigation, evidence for such an association was evaluated in Chesapeake Bay and other mid-Atlantic estuaries (1999–2001). A statistical approach was used, without invoking causality, to test whether there is an association between the prevalence of externally-visible lesions in fish populations above background levels and the presence of Pfiesteria spp. in co-located water and fish samples. Externally visible anomalies (e.g. ulcers, necrosis, parasites, etc.) were recorded for Atlantic menhaden (Brevoortia tyrannus) and all other fish collected. Polymerase chain reaction (PCR) techniques were used to test for the presence of Pfiesteria spp. in water samples collected at routine and rapid response sampling events. No actively toxic Pfiesteria was found during this study. Fine-scale (within a given sample site) and broad-scale (estuary-wide sampling) comparisons showed positive associations between externally-visible fish lesions in menhaden populations and the presence of Pfiesteria spp. in co-located samples. Logistic regression modeling of Pfiesteria detection probabilities as a function of prevalence of menhaden with lesions was significant (P = 0.0096). Reductions in the false positive (tests indicating Pfiesteria presence when its absent) and false negative (tests indicating Pfiesteria is absent when it is actually present) rates occurred when the minimum sample size threshold increased from 1 to 30 fish (P = 0.003–0.001). This association served as a useful field indicator of potential HAB activity that could warrant further field investigation and testing.     

Modeling the influence of nutrients, turbulence and grazing on Pfiesteria population dynamics

A semi-idealized marine ecosystem model, designed as a heuristic tool for exploring the population dynamics of non-inducible versus toxic forms of Pfiesteria is described. The model is based on empirical evidence suggesting that these differing functional types of Pfiesteria also differ substantially in terms of what they eat and how they utilize it to optimize their growth. Non-inducible strains are similar to other mixotrophic dinoflagellates, whereas toxic strains may consume organic matter and detritus, produce toxins and attack fish. In our model formulation we represent these differences in a simplified way: the non-inducible strain is kleptochloroplastidic and it can take up DIN, but it cannot utilize DON, whereas the toxic strain is heterotrophic, it cannot utilize DIN, but it can utilize DON directly. These differences give rise to very different impacts on prey and nutrient concentrations in our model. Under high DIN/DON ratio conditions, the non-inducible cells grew much faster and were therefore more likely to bloom, but this advantage is substantially mitigated when the DIN/DON ratio is low. A turbulence parameterization was also incorporated into our model. The effect of this was to reduce the grazing rate of Pfiesteria when turbulence levels are high. According to our model, increased turbulence is more detrimental to the toxic functional type because it grows more slowly. The further imposition of microzooplankton grazing in the model showed that top-down control effects can be very significant, which is consistent with both laboratory and field studies and the general idea that plankton blooms can only happen in the absence of substantial grazing control. In general, our model results suggest that non-toxic blooms are more likely to occur in more turbulent inorganic-nutrient rich conditions, which are often found in more open coastal and estuarine waters that are subject to high inorganic loading. In contrast, toxic blooms are more likely to occur in calm, organic-nutrient rich conditions, which are often found in shallow, protected tributaries that are subject to high organic nutrient loading. Our model results also support the idea that the absence of strong grazing pressure is a prerequisite to bloom formation for both non-inducible and toxic strains of Pfiesteria. These results are generally consistent with observed patterns of toxic Pfiesteria blooms in Chesapeake Bay, the Neuse River of North Carolina and many other coastal and estuarine environments.     

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

Biology of Lilioceris spp. (Coleoptera: Chrysomelidae) and their parasitoids in Europe

The biology and parasitoid complex of the lily leaf beetle (LLB), Lilioceris lilii Scopoli, and two congeneric species were investigated in Europe, as part of a biological control program against the LLB in North America. Eggs, larvae, and adults of L. lilii were collected in several countries in Europe, on both cultivated and wild Lilium spp., and reared in the laboratory and under natural conditions. Parasitoids were obtained and their biologies were studied. Similar investigations were made in Switzerland on two closely related species Lilioceris tibialis (Villa) found on wild Lilium spp., and Lilioceris merdigera (L.) on several other Liliaceae. The three species are strictly univoltine. Adults overwinter and lay eggs on leaves in early spring. The three beetle species have four instars, which were characterized by their head capsule width. Pupation occurs in a cocoon in the soil. Adults emerge in late summer and start feeding before reaching overwintering sites. Egg and larval parasitoids were obtained. Eggs of L. lilii and L. merdigera were parasitized by the mymarid Anaphes sp., a multivoltine species that needs alternate hosts for overwintering. Larvae were heavily attacked by several parasitoids, among which the most abundant were three ichneumonids, Lemophagus pulcher (Szepligeti), L. errabundus (Gravenhorst), and Diaparsis jucunda (Holmgren), and the eulophid Tetrastichus setifer Thomson. All four parasitoid species were found in the three beetles and in most European regions, but strong variations were observed in their relative abundance among hosts and geographic regions. Three of the four main larval parasitoids are strictly univoltine, whereas L. pulcher has a partial second generation. Lemophagus spp. are frequently parasitized by the ichneumonid hyperparasitoid Mesochorus lilioceriphilus Schwenke. Further details of the biology of the parasitoids are described, and their potential as biological control agents is discussed.     

Fruit maturation patterns of Carya spp. (Juglandaceae): an intra-crown analysis of growth and reproduction

Summary Fruit survival patterns, from fertilization to maturation, were examined for Carya ovata and C. tomentosa in a New Jersey USA forest. We observed fruiting and shoot growth characteristics over a 3-yr period to determine: (1) the patterns of fruit survivorship (from initiation to maturity) within and among years, (2) the relationships between shoot growth, fruit initiation, and fruit survival to maturity, and (3) the influence of phytophagous insects on fruit survival. We found that within years, smaller infructescences (1–2 fruits) exhibited greater relative survivorship than larger ones (3–4 fruits); however, absolute nut production was greatest for mid-sized infructescences (2–3 fruits). Among years, fruit survivorship varied considerably within populations. Across the 3-yr period we observed average fruit survivorship to be convex, linear, and concave, respectively. Likewise, shoot characteristics (length, width, number of leaves) varied concomitantly (decreasing fruit survivorship was accompanied by decreasing shoot length and number of leaves). Within years, we found no strong relationship between shoot characteristics and infructescence size and survival. The patterns of tree-to-tree variation suggested a strong genetic basis to shoot growth and fruit maturation. However, patterns of variation within and among years also indicated a strong environmental influence on these traits as well. Natural phytophagy by insects was observed to be low (<5%); however, shoot defoliations of 10–25% were not uncommon. Experimental defoliations (ambient, 10–15%, 20–40%, and 75–100%) did not result in reduced survival to maturity. Collectively, the data suggest that year-to-year variability in shoot growth has a greater influence on fruit maturation patterns than within year fruit-shoot relations.     

First report of Alexandrium taylori and Alexandrium peruvianum (Dinophyceae) in Malaysia waters

The occurrence of Alexandrium taylori and Alexandrium peruvianum is reported for the first time in Malaysia waters. The Malaysian A. taylori isolates were pyriform in shape with a transdiameter range of 36–40 μm and a cell length range of 33–37 μm. The first apical plate (1′) was pentagonal with two distinctive anterior margins. No direct connection between 1′ and the apical pore complex was observed. The posterior sulcal plate (S.p.) was large, elongated and oblique to the right with anterior projections. The ventral pore (vp) was relatively large and situated at a confluence point of 1′, the second apical (2′) and the fourth apical (4′) plates. Cells of A. peruvianum were slightly anteriorly and posteriorly compressed. S.p. had an irregular pentagonal shape, with the anterior margin divided into 2 portions. 1′ was boomerang-shaped with a large and truncated ventral pore in the middle right margin. The anterior right margin of 1′ was straight. The sixth precingular plate (6″) was wider than long. The anterior sulcal plate (S.a.) was triangular and lacked a left portion extension. In laboratory cultures, both A. taylori and A. peruvianum produced paralytic shellfish toxins, with GTX4 and GTX6 as the predominant toxin, respectively. This is the first report of PSP toxins production for both species as well as the occurrences in Malaysia waters.     

Open field host selection and behavior by tamarisk beetles (Diorhabda spp.) (Coleoptera: Chrysomelidae) in biological control of exotic saltcedars (Tamarix spp.) and risks to non-target athel (T. aphylla) and native Frankenia spp.

Biological control of invasive saltcedars (Tamarix spp.) in the western U.S. by exotic tamarisk leaf beetles, Diorhabda spp., first released in 2001 after 15 years of development, has been successful. In Texas, beetles from Crete, Greece were first released in 2004 and are providing control. However, adults alight, feed and oviposit on athel (Tamarix aphylla), an evergreen tree used for shade and as a windbreak in the southwestern U.S. and México, and occasionally feed on native Frankenia spp. plants. The ability of tamarisk beetles to establish on these potential field hosts was investigated in the field. In no-choice tests in bagged branches, beetle species from Crete and Sfax, Tunisia produced 30–45% as many egg masses and 40–60% as many larvae on athel as on saltcedar. In uncaged choice tests in south Texas, adult, egg mass and larval densities were 10-fold higher on saltcedar than on adjacent athel trees after 2 weeks, and damage by the beetles was 2- to 10-fold greater on saltcedar. At a site near Big Spring, in west-central Texas, adults, egg masses and 1st and 2nd instar larvae were 2- to 8-fold more abundant on saltcedar than on athel planted within a mature saltcedar stand being defoliated by Crete beetles, and beetles were 200-fold or less abundant or not found at all on Frankenia. At a site near Lovelock, Nevada, damage by beetles of a species collected from Fukang, China was 12–78% higher on saltcedar than on athel planted among mature saltcedar trees undergoing defoliation. The results demonstrate that 50–90% reduced oviposition on athel and beetle dispersal patterns within resident saltcedar limit the ability of Diorhabda spp. to establish populations and have impact on athel in the field.     

Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

Suitability of Aphis fabae, Myzus persicae (Homoptera: Aphididae) and Aleyrodes proletella (Homoptera: Aleyrodidae) as prey for Coccinella undecimpunctata (Coleoptera: Coccinellidae)

The suitability of Aphis fabae Scopoli, Myzus persicae Sulzer and Aleyrodes proletella L. as food sources for Coccinella undecimpunctata L. was evaluated by studying the impact of prey consumption on the predator’s population growth parameters and feeding parameters. Unlike A. proletella, A. fabae and M. persicae supported the development and reproduction of C. undecimpunctata. A. fabae and M. persicae were considered to be essential prey, whereas A. proletella was considered to be an alternative prey. Aphid species showed different degrees of suitability: M. persicae significantly decreased the pre-oviposition period and increased adult longevity, fecundity and fertility compared with A. fabae. Moreover, A. fabae represents a suitable diet for larval development, but is not a suitable food source for adult reproduction. The predator’s population growth parameters, R₀, r_m and λ were increased with M. persicae, whereas T decreased. We found that the 4th instar larvae were the most voracious, particularly when fed on M. persicae; nevertheless, with this prey daily weight gain and feeding efficiency of 4th instar larvae were similar to that of individuals fed with A. fabae.     

Morphology and taxonomy of chain-forming species of the genus Cochlodinium (Dinophyceae)

The morphology of an unarmored chain-forming harmful dinoflagellate Cochlodinium polykrikoides and its similar species such as Cochlodinium catenatum, Cochlodinium fulvescens, and Cochlodinium convolutum was carefully observed, emphasizing the single cell stage for clarifying taxonomically important morphological features. To differentiate C. polykrikoides from C. convolutum, the shape and the position of the nucleus are useful characters. C. polykrikoides also differs from C. fulvescens in being smaller in size, possessing many rod-shaped chloroplasts and having the sulcus running just below the cingulum on the dorsal surface. Careful observation of the ichnotype of C. catenatum suggests that C. catenatum sensu Kofoid and Swezy collected from off La Jolla, CA, USA, is not identical to C. catenatum sensu Okamura and is probably a different species, in having no chloroplasts and a nucleus positioned at the center of the cell. In addition, C. polykrikoides has many morphological features in common with C. catenatum sensu Okamura except for slightly elongate cells and is probably a junior synonym of this species.     

Leptodiscaceans (Noctilucales, Dinophyceae) from the Pacific Ocean: First records of Petalodinium and Leptodiscus beyond the Mediterranean Sea

Records of dinoflagellates of the family Leptodiscaceae (Noctilucales) from the Kuroshio Current, Philippine, Celebes, Sulu, South China Seas and the western and central Equatorial Pacific Ocean are described. Scaphodinium mirabile was the most common leptodiscacean. Two specimens that differed from the type species of Scaphodinium were found: one specimen showed a highly bifurcate proximal extremity and another showed two dissimilar proboscides from the distal extremity. Another unidentified leptodiscacean showed an arrowhead-shaped contour with the margins folded. Six specimens of Petalodinium porcelio were found, being the first record beyond the Mediterranean-Black Seas. Six specimens were tentatively assigned to the genus Leptodiscus, being the first record beyond the western Mediterranean Sea. The folded specimens that ranged from 90 to 120 μm in diameter and with a prominent flagellum were tentatively considered to be young specimens of Leptodiscus. The abundance of the leptodiscaceans is underestimated in the world's oceans.     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

Yeast Srp1, a nuclear protein related toDrosophila and mouse pendulin, is required for normal migration, division, and integrity of nuclei during mitosis

This paper describes genes from yeast and mouse with significant sequence similarities to aDrosophila gene that encodes the blood cell tumor suppressor pendulin. The protein encoded by the yeast gene, Srp1p, and mouse pendulin share 42% and 51% amino acid identity withDrosophila pendulin, respectively. All three proteins consist of 10.5 degenerate tandem repeats of 42 amino acids each. Similar repeats occur in a superfamily of proteins that includes theDrosophila Armadillo protein. All three proteins contain a consensus sequence for a bipartite nuclear localization signal (NLS) in the N-terminal domain, which is not part of the repeat structure. Confocal microscopic analysis of yeast cells stained with antibodies against Srp1p reveals that this protein is intranuclear throughout the cell cycle. Targeted gene disruption shows thatSRP1 is an essential gene. Despite their sequence similarities,Drosophila and mouse pendulin are unable to rescue the lethality of anSRP1 disruption. We demonstrate that yeast cells depleted of Srp1p arrest in mitosis with a G2 content of DNA. Arrested cells display abnormal structures and orientations of the mitotic spindles, aberrant segregation of the chromatin and the nuclei, and threads of chromatin emanating from the bulk of nuclear DNA. This phenotype suggests that Srplp is required for the normal function of microtubules and the spindle pole bodies, as well as for nuclear integrity. We suggest that Srp1p interacts with multiple components of the cell nucleus that are required for mitosis and discuss its functional similarities to, and differences fromDrosophila pendulin.     

Hyperparasitism by Mesochorus spp. (Hymenoptera: Ichneumonidae) in Peristenus sp. (Hymenoptera: Braconidae) and development of PCR primers for hyperparasitoid detection

Peristenus sp. pupae collected from Lygus spp. nymphs in 2001 and 2002 were over-wintered in the laboratory. In both years, more than 30% of adults emerging from over-wintering pupae were identified as ichneumonid hyperparasitoids, Mesochorus curvulus Thomson and Meschorus sp. (Hymenoptera: Ichneumonidae). At the end of the over-wintering period, Peristenus sp. males emerged first followed by Peristenus sp. females and finally Mesochorus spp. The male:female ratio in emerging Peristenus sp. adults was skewed towards males. The Internal Transcribed Spacer (ITS) region and the cytochrome oxidase I (COI) gene from Mesochorus spp. were sequenced. ITS sequences were used to develop PCR primers to detect Mesochorus spp. hyperparasitism in the primary host, Lygus spp. PCR analysis of field-collected Lygus spp. nymphs gave similar estimates of Mesochorus spp. hyperparasitism to the rearing protocols (25–28%). Sequence analysis of COI and ITS regions and subsequent restriction endonuclease analysis of ITS PCR products from Mesochorus spp. indicate the presence of two genotypes in the population. The possibility that these two genotypes represent separate or cyrptic species is discussed.