Naturally Occurring Yeasts Associated with Thaumatotibia leucotreta Can Enhance the Efficacy of the Cryptophlebia Leucotreta Granulovirus.

Yeasts associated with lepidopteran pests have been shown to play a role in their survival, development, and oviposition preference. It has been demonstrated that combining these yeasts with existing biological control agents can enhance their efficacy. The tortricid Thaumatotibia leucotreta is a phytosanitary pest in the South African citrus industry, with the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV) being one of the components that can control this pest. Several yeast species were shown to be associated with T. leucotreta larvae, which affected their behaviour and development. A series of detached fruit bioassays were performed to determine whether the combination of yeast with CrleGV enhances its efficacy. These assays included determining the optimal yeast/virus ratio, testing all isolated yeast species in combination with CrleGV, and further improving yeast/virus formulation by adding an adjuvant. The optimal yeast concentration to use alongside CrleGV was determined to be 106 cells·mL-1. Pichia kluyveri, P. kudriavzevii, Kluyveromyces marxianus, and Saccharomyces cerevisiae in combination with CrleGV reduced larval survival compared to CrleGV alone. The addition of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae in combination with CrleGV did not notably improve their effectiveness; however, there was an observed decrease in larval survival. In future studies, field trials will be conducted with combinations of CrleGV and P. kudriavzevii or S. cerevisiae to investigate whether these laboratory findings can be replicated in orchard conditions.

Disentangling thermal effects using life cycle simulation modelling on the biology and demographic parameters of Dolichogenidea gelechiidivoris, a parasitoid of Tuta absoluta.

Dolichogenidea gelechiidivoris (Marsh) (Syn. Apanteles gelechiidivoris) (Hymenoptera: Braconidae), a konoibiont larval endoparasitoid of the South American tomato pinworm Tuta absoluta (Meyrick), was imported into Kenya in 2017 for the first classical biological control of T. absoluta in Africa. We determined the thermal thresholds of D. gelechiidivoris, with T. absoluta as host, using life cycle simulation modelling. Life-table data of D. gelechiidivoris were generated at six constant temperatures (10, 15, 20, 25, 30, and 35 °C, 65 ± 5% RH, and 12L: 12D photoperiod). Multiple non-linear functions were fitted to model some aspects of the biology of the parasitoid, including its longevity, mortality, reproduction, and senescence using Insect Life Cycle Modelling (ILCYM) software. The phenology models established were used to estimate life table parameters. Except at 35 °C, D. gelechiidivoris completed its development (i.e., egg-larval-pupa-adult) at all tested temperatures. The minimum temperature threshold from egg to cocoon was 7.0 °C and 5.9 °C from cocoon to adult wasp, while the maximum temperature thresholds were 34 °C and 36 °C, respectively. The optimal temperature for immature survival was 20-25 °C, while fecundity was optimum at 22.5 °C, with 69.5 eggs per female. Simulations of the population growth parameters indicated that the intrinsic rate of increase (rm) was maximum at 20 °C with 15 daughters per female per generation. Based on our findings the release of D. gelechiidivoris for inoculative augmentation in countries with temperatures ranging between 15 and 30 °C could be considered to control the pest.

The benefits to sub-Saharan Africa of the biological control of weeds: already considerable, but could be far greater.

Sub-Saharan Africa (SSA) is climatically diverse, with many biomes. Many species of invasive alien plants (IAPs) are present, with severe negative impacts that largely remain unquantified. Importation biological weed control (IBWC) has been practiced on the continent since the early 20th century, with some notable successes. Weed biocontrol agents (WBA) (141 species) have been released in 30 countries on 69 weed species, and spread to a further eight countries. South Africa has the most active IBWC program, while several projects are underway in other countries, involving either the release of WBA or monitoring of their spread and impact. However, given the large and increasing footprint of IAPs, and the poor prospects for their successful management using other control methods, we discuss reasons for the relatively low uptake of IBWC in SSA, and suggest ways to increase this.

Mutualism between Gut-Borne Yeasts and Their Host, Thaumatotibia leucotreta, and Potential Usefulness in Pest Management

Thaumatotibia leucotreta is endemic to southern Africa and is highly significant for various fruit industries, including the South African citrus industry, due to its classification as a phytosanitary pest. Mutualistic associations between C. pomonella, closely related to T. leucotreta, and yeasts have previously been described and reported to reduce larval mortality and enhance larval development. Here, we determined which yeast species occur naturally in the gut of T. leucotreta larvae and investigated whether any of the isolated yeast species affect their behaviour and development. Navel oranges infested with T. leucotreta larvae were collected from geographically distinct provinces in South Africa, and the larvae were processed for analysis of naturally occurring associated yeasts. Six yeast species were isolated and identified from the guts of these T. leucotreta larvae via PCR amplification and sequencing of the ITS region of rDNA and D1/D2 domain of large ribosomal subunit. Larval development and attraction assays were conducted, and T. leucotreta larvae that fed on Navel oranges inoculated with yeast had accelerated developmental periods and reduced mortality rates. Neonate T. leucotreta were also attracted to YPD broth cultures inoculated with yeast for feeding. Oviposition preference assays were conducted with adult T. leucotreta females. Navel oranges inoculated with yeast were shown to influence the oviposition preference of adult females. Yeasts harbour the potential for use in biocontrol, especially when combined with other well-established control methods. This study provides a platform for future research into incorporating yeast with current biological control agents as a novel option for controlling T. leucotreta in the field.

Heavy Metals Assimilation by Native and Non-Native Aquatic Macrophyte Species: A Case Study of a River in the Eastern Cape Province of South Africa.

There is continuous deterioration of freshwater systems globally due to excessive anthropogenic inputs, which severely affect important socio-economic and ecological services. We investigated the water and sediment quality at 10 sites along the severely modified Swartkops River system in the Eastern Cape Province of South Africa and then quantified the phytoremediation potential by native and non-native macrophyte species over a period of 6 months. We hypothesized that the presence of semi and permanent native and non-native macrophytes mats would reduce water and sediment contamination through assimilation downriver. Our results were variable and, thus, inconsistent with our hypotheses; there were no clear trends in water and sediment quality improvement along the Swartkops River. Although variable, the free-floating non-native macrophyte, Pontederia (=Eichhornia) crassipes recorded the highest assimilation potential of heavy metals in water (e.g., Fe and Cu) and sediments (e.g., Fe and Zn), followed by a submerged native macrophyte, Stuckenia pectinatus, and three native emergent species, Typha capensis, Cyperus sexangularis, and Phragmites australis. Pollution indices clearly showed the promising assimilation by native and non-native macrophytes species; however, the Swartkops River was heavily influenced by multiple non-point sources along the system, compromising the assimilation effect. Furthermore, we emphasise that excessive anthropogenic inputs compromise the system's ability to assimilate heavy metals inputs leading to water quality deterioration.

Can the Combined Use of the Mirid Predator Nesidiocoris tenuis and a Braconid Larval Endoparasitoid Dolichogenidea gelechiidivoris Improve the Biological Control of Tuta absoluta?

The koinobiont solitary larval endoparasitoid Dolichogenidea gelechiidivoris (Marsh) (Syn.: Apanteles gelechiidivoris) (Hymenoptera: Braconidae) and the predatory bug Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) are important natural enemies of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), a serious pest of tomato. Although N. tenuis preferentially feeds on T.absoluta eggs, it is also recorded as a predator of first and second instar larval stages. Dolichogenidea gelechiidivoris preferentially seeks these early larval stages of T. absoluta for oviposition. The occurrence of intraguild predation between N. tenuis and D. gelechiidivoris and the consequences on the oviposition performance of D. gelechiidivoris were investigated in the laboratory. Regardless of the manner of introduction (i.e., the sequence of combinations with D. gelechiidivoris) or density (i.e., number of N. tenuis combined with D. gelechiidivoris), the presence of N. tenuis did not affect the oviposition performance of D. gelechiidivoris or the parasitoid's progeny. Combination assays revealed that the efficacy of the combined use of N. tenuis and D. gelechiidivoris in controlling T. absoluta populations was significantly higher than that of either natural enemy alone. Our results highlight the potential of combining mirid predators and koinobiont larval endoparasitoids to control T. absoluta. The findings further contribute to data supporting the release of D. gelechiidivoris in tomato agroecosystems for the control of T. absoluta in Africa, where N. tenuis is widespread and abundant.

Mind the gap: the delayed recovery of a population of the biological control agent Megamelus scutellaris Berg. (Hemiptera: Delphacidae) on water hyacinth after winter.

Cold winter temperatures significantly affect the biological control effort against water hyacinth, Pontederia ( = Eichhornia) crassipes Mart. (Pontederiaceae), in more temperate regions around the world. The population dynamics of the planthopper Megamelus scutellaris Berg. (Hemiptera: Delphacidae), a newly released biological control agent of water hyacinth, were recorded on the Kubusi River in the Eastern Cape Province (South Africa) over 15 months to determine the population recovery post-winter. Megamelus scutellaris incurred a severe population decline at the onset of winter when the water hyacinth plants became frost damaged. The combined effect of a population bottleneck and low minimum winter temperatures (6.12°C) below the agent's lower developmental threshold (11.46°C) caused a post-winter lag in agent density increase. Subsequently, the maximum agent population density was only reached at the end of the following summer growing season which allowed the water hyacinth population to recover in the absence of any significant biological control immediately post-winter. Supplementary releases of agents from mass-reared cultures at the beginning of the growing season (spring) is suggested as a potential method of reducing the lag-period in field populations in colder areas where natural population recovery of agents is slower.

Additive interaction between a root-knot nematode Meloidogyne javanica and a root-feeding flea beetle Longitarsus bethae on their host Lantana camara.

BACKGROUND: Changes in plants induced by one species have indirect effects on interactions with other species, thus shaping their abundances. The root-feeding beetle Longitarsus bethae released as a biological control agent for Lantana camara has established at a few sites in South Africa. Lantana camara plants infected with the root-knot nematode Meloidogyne javanica often exhibit an increase in L. bethae abundance, suggesting that nematode-infected plants could be enhancing the development of the beetle. This study investigated the interaction between L. bethae and M. javanica. RESULTS: The study showed that galling by M. javanica occurred at the highest inoculation of 300 eggs of L. bethae per plant. Longitarsus bethae performed over twofold better on M. javanica-infected L. camara roots compared with healthy roots, and 275 adult L. bethae progeny, with slightly larger body size, emerged from M. javanica-infected compared with 167 adults that emerged from healthy plants. Fresh gall weight from treatments where both L. bethae and M. javanica were combined was 36% higher than that from M. javanica only, suggesting that the combination of both species induces more galling than the nematode does alone. The total biomass of plants with M. javanica only, L. bethae only, and the two species combined was reduced by 48%, 24% and 50%, respectively. CONCLUSION: Meloidogyne javanica improves the performance of L. bethae, and combination of the two species has an additive negative effect on L. camara. The interaction between M. javanica and L. bethae could enhance the biological control of L. camara. © 2019 Society of Chemical Industry.

A new approach to the biological monitoring of freshwater systems: Mapping nutrient loading in two South African rivers, a case study.

Excessive addition of nitrogen (N) has threatened aquatic ecosystems for decades. Traditional water quality and biological monitoring assessment tools are widely used for monitoring nutrient loads and ecosystem health, but most of these methods cannot distinguish between different types and sources of pollution. This is a challenge, particularly when dealing with non-point sources of anthropogenic nitrogen inputs into freshwater systems. Recent laboratory studies using stable isotopic ratios (δ15N and C/N) of aquatic macrophytes (duckweed: Spirodela spp.) have shown successful differentiation and mapping between different N-sources and further, showed abilities to act as early warning indicators for environmental N-loading. Therefore, the aim of this study was to field test the potential of stable isotopic values of transplanted Spirodela spp. to map temporal and spatial N-loading variation and determine the main sources of N-loading in two river systems in the Eastern Cape Province of South Africa, using previously grown, isotopically calibrated and transplanted Spirodela plants, collected over a 13-month sampling period. Nitrogen isotopic values (δ15N) of Spirodela plants traced environmental N-loading and identified pollution hotspots and sources through time and space over a wide range of nutrient gradients. δ15N isotopic values of Spirodela spp. provided detailed dynamics on N-loading, therefore supporting its utilisation in the biological monitoring of ecosystem health and the early detection of eutrophication in freshwater systems.

The South America Tomato Leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), Spreads Its Wings in Eastern Africa: Distribution and Socioeconomic Impacts.

Following the arrival of Tuta absoluta Meyrick in the eastern African subregion in 2012, several studies have shown numerous ecological aspects of its invasion. We investigated the impact of T. absoluta on people's livelihoods across four counties of Kenya. Here, 200 farmers in the country were interviewed in person using semistructured questionnaires. In addition to livelihood surveys, T. absoluta distribution was mapped between 2016 and 2018 to determine its current distribution across four countries (Kenya, Sudan, Tanzania, and Uganda) in the subregion. Albeit a recent invader, T. absoluta is abundant and distributed throughout the subregion and is viewed as the worst invasive alien species of agriculturally sustainable livelihoods by tomato farmers. The arrival of T. absoluta in the subregion has resulted in livelihood losses and increased both the cost of tomato production and frequency of pesticide application. We recommend the implementation of biological control along, with other control measures in an integrated approach, against T. absoluta in the subregion, where its impact on sustainable livelihoods is serious and long-term control strategies are required to curb its detrimental effects.

Genome Analysis of A Novel South African Cydia pomonella granulovirus (CpGV-SA) with Resistance-Breaking Potential.

The complete genome of an endemic South African Cydia pomonella granulovirus isolate was sequenced and analyzed. Several missing or truncated open reading frames (ORFs) were identified, including a 24 bp deletion in the pe38 gene which is reported to be associated with type I resistance-breaking potential. Comparison of single nucleotide polymorphisms (SNPs) with five other fully sequenced CpGV isolates identified 67 unique events, 47 of which occurred within ORFs, leading to several amino acid changes. Further analysis of single nucleotide variations (SNVs) within CpGV-SA revealed that this isolate consists of mixed genotypes. Phylogenetic analysis using complete genome sequences placed CpGV-SA basal to M, I12 and E2 and distal to S and I07 but with no distinct classification into any of the previously defined CpGV genogroups. These results suggest that CpGV-SA is a novel and genetically distinct isolate with significant potential as a biopesticide for management of codling moth (CM), not only in South Africa, but potentially in other pome fruit producing countries, particularly where CM resistance to CpGV has been reported.

Cryptophlebia peltastica Nucleopolyhedrovirus Is Highly Infectious to Codling Moth Larvae and Cells.

Cydia pomonella granulovirus (CpGV) is a cornerstone of codling moth (Cydia pomonella) control in integrated and organic pome fruit production, though different types of resistance to CpGV products have been recorded in codling moth field populations in Europe for several years. Recently, a novel baculovirus named Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) was isolated from a laboratory culture of the litchi moth, Cryptophlebia peltastica, in South Africa. Along with CpGV, it is the third known baculovirus that is infectious to codling moth. In the present study, parameters of infectiveness of CrpeNPV, such as the median lethal concentration and median survival time, were determined for codling moth larvae susceptible or resistant to CpGV. In addition, the permissiveness of a codling moth cell line with respect to infection by CrpeNPV budded virus was demonstrated by infection and gene expression studies designed to investigate the complete replication cycle. Investigations of the high degree of virulence of CrpeNPV for codling moth larvae and cells are of high significant scientific and economic value and may offer new strategies for the biological control of susceptible and resistant populations of codling moth.IMPORTANCE The emergence of codling moth populations resistant to commercially applied isolates of CpGV is posing an imminent threat to organic pome fruit production. Very few CpGV isolates are left that are able to overcome the reported types of resistance, emphasizing the demand for new and highly virulent baculoviruses. Here we report the recently discovered CrpeNPV as highly infectious to all types of resistant codling moth populations with a high speed of killing, making it a promising candidate baculovirus in fighting the spread of resistant codling moth populations.

Development of a Postharvest Cold Treatment for Cryptophlebia peltastica (Lepidoptera: Tortricidae) for Export of Litchis From South Africa.

The litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae), is endemic to sub-Saharan Africa and certain Indian Ocean islands. It is an important pest of litchis and to a lesser extent macadamias. Litchis are exported to certain markets that consider C. peltastica as a phytosanitary pest. Consequently, an effective postharvest phytosanitary treatment is required. This study sought to develop a cold disinfestation treatment for this purpose. First, it was established that the fifth instar was the most cold-tolerant larval stage, as it was the only instar for which there was still some survival after 12 d at 1°C. It was then determined that cold treatment trials could be conducted in artificial diet, as there was no survival of fifth instar C. peltastica in litchis after only 9 d at 1°C, whereas it took 15 d at this temperature before no survival of fifth instar C. peltastica was recorded in artificial diet. Consequently, cold susceptibility of fifth instar C. peltastica and the most cold-tolerant larval stages (fourth and fifth instar) of false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), were compared in artificial diet. There was no survival of C. peltastica after 13 d at 1°C, whereas this was only so for T. leucotreta after 16 d. Consequently, it can be concluded that any cold treatment that has been proven effective against T. leucotreta would be as effective against C. peltastica. Finally, it was confirmed that the cold susceptibility of T. leucotreta in artificial diet did not overestimate the effect of cold on T. leucotreta larvae in litchis.

Morphological, genetic and biological characterisation of a novel alphabaculovirus isolated from Cryptophlebia peltastica (Lepidoptera: Tortricidae).

Cryptophlebia peltastica is an agricultural pest of litchis and macadamias in South Africa with phytosanitary status for certain markets. Current control methods rely on chemical, cultural and classical biological control. However, a microbial control option has not been developed. An Alphabaculovirus from C. peltastica was recovered from a laboratory reared colony and morphologically characterised by transmission electron microscopy (TEM). Analysis of occlusion bodies indicated a single NPV (SNPV) varying in size from 421 to 1263 nm. PCR amplification and sequencing of the polh gene region using universal primers followed by BLAST analysis revealed a 93% similarity to a partial polh gene sequence from Epinotia granitalis NPV. Further genetic characterisation involving single restriction endonuclease (REN) digestion of genomic DNA was carried out to generate profiles for comparison against other baculovirus species and potential new isolates of the same virus. The complete genome of the virus was sequenced, assembled and analysed for a more comprehensive genetic analysis. The genome was 115728 base pairs (bp) in length with a GC content of 37.2%. A total of 126 open reading frames (ORFs) were identified with minimal overlap and no preference in orientation. Bioassays were used to determine the virulence of the NPV against C. peltastica. The NPV was virulent against C. peltastica with an LC50 value of 6.46 × 103 OBs/ml and an LC90 value of 2.46 × 105 OBs/ml, and time mortality ranging between 76.32 h and 93.49 h. This is the first study to describe the isolation and genetic characterisation of a novel SNPV from C. peltastica, which has potential for development into a biopesticide for the control of this pest in South Africa.

Expansive reed populations-alien invasion or disturbed wetlands?

The tall-statured grasses in the genus Phragmites are dominant vegetation in wetlands worldwide and thus play a vital role in ecosystem functioning. As a result, Phragmites spp. are some of the most widely studied plants; particularly in areas where changes to their abundances have occurred, most notably in Europe and North America. In southern Africa a pattern of reed expansion has occurred in recent decades that has shown a similar trend to cryptic invasions reported in North America. This study used molecular techniques to explore the phylogeography of P. australis and P. mauritianus in the region to investigate whether the expansion is due to an alien invasion or local factors such as wetland disturbance. Three haplotypes were found and all haplotypes are presently considered African haplotypes (haplotype K for P. australis and haplotype V and AP for P. mauritianus). Both Phragmites spp. were found to have high genetic diversity. Microsatellite and grass-waxy analysis also found evidence of hybridization between the two species. No evidence was found for a recent cryptic invasion of non-native haplotypes in southern Africa. The expansion of P. australis and P. mauritianus is therefore most likely a result of anthropogenic activity. Identifying and mitigating the human-mediated factors that may be contributing to reed growth, such as eutrophication and sedimentation, should be the focus of future management protocols.

Developmental and reproductive performance of a specialist herbivore depend on seasonality of, and light conditions experienced by, the host plant.

Host plant phenology (as influenced by seasonality) and light-mediated changes in the phenotypic and phytochemical properties of leaves have been hypothesised to equivocally influence insect herbivore performance. Here, we examined the effects of seasonality, through host plant phenology (late growth-season = autumn vs flowering-season = winter) and light environment (shade vs full-sun habitat) on the leaf characteristics of the invasive alien plant, Chromolaena odorata. In addition, the performance of a specialist folivore, Pareuchaetes insulata, feeding on leaves obtained from both shaded and full-sun habitats during autumn and winter, was evaluated over two generations. Foliar nitrogen and magnesium contents were generally higher in shaded plants with much higher levels during winter. Leaf water content was higher in shaded and in autumn plants. Total non-structural carbohydrate (TNC) and phosphorus contents did not differ as a function of season, but were higher in shaded foliage compared to full-sun leaves. Leaf toughness was noticeably higher on plants growing in full-sun during winter. With the exception of shaded leaves in autumn that supported the best performance [fastest development, heaviest pupal mass, and highest growth rate and Host Suitability Index (HSI) score], full-sun foliage in autumn surprisingly also supported an improved performance of the moth compared to shaded or full-sun leaves in winter. Our findings suggest that shaded and autumn foliage are nutritionally more suitable for the growth and reproduction of P. insulata. However, the heavier pupal mass, increased number of eggs and higher HSI score in individuals that fed on full-sun foliage in autumn compared to their counterparts that fed on shaded or full-sun foliage in winter suggest that full-sun foliage during autumn is also a suitable food source for larvae of the moth. In sum, our study demonstrates that seasonal and light-modulated changes in leaf characteristics can affect insect folivore performance in ways that are not linear.

Genome Analysis and Genetic Stability of the Cryptophlebia leucotreta Granulovirus (CrleGV-SA) after 15 Years of Commercial Use as a Biopesticide.

Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is an indigenous pest in southern Africa which attacks citrus fruits and other crops. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme incorporating the baculovirus Cryptophlebialeucotreta granulovirus (CrleGV-SA) as a biopesticide has been implemented. This study investigated the genetic stability of a commercially produced CrleGV-SA product that has been applied in the field since 2000. Seven representative full-genome sequences of the CrleGV-SA isolate spanning a 15-year period were generated and compared with one another. Several open reading frames (ORFs) were identified to have acquired single nucleotide polymorphisms (SNPs) during the 15-year period, with three patterns observed and referred to as "stable", "reversion", and "unstable switching". Three insertion events were also identified, two of which occurred within ORFs. Pairwise multiple alignments of these sequences showed an identity ranging from 99.98% to 99.99%. Concentration-response bioassays comparing samples of CrleGV-SA from 2000 and 2015 showed an increase in virulence toward neonate T. leucotreta larvae. The CrleGV-SA genome sequence generated from the 2015 sample was compared to the Cape Verde reference genome, CrleGV-CV3. Several fusion events were identified between ORFs within these genomes. These sequences shared 96.7% pairwise identity, confirming that CrleGV-SA is a genetically distinct isolate. The results of this study indicate that the genome of CrleGV-SA has remained stable over many years, with implications for its continued use as a biopesticide in the field. Furthermore, the study describes the first complete baculovirus genome to be sequenced with the MinION (Oxford Nanopore, Oxford, UK) platform and the first complete genome sequence of the South African CrleGV isolate.

A new species of Phanerotoma Wesmael (Hymenoptera: Braconidae: Cheloninae) parasitoid of the carob moth in South Africa.

A new species, Phanerotoma carobivora van Achterberg & Thackeray, sp. nov. is described from South Africa. It is a common endoparasitoid of the carob moth (Ectomyelois ceratoniae Zeller (Pyralidae) on pecan (Carya illinoinensis (Wangenh.) K. Koch) and citrus fruits in South Africa. Mean percentage of parasitism varied 2-30% between host plants and sampled localities.

Two in one: cryptic species discovered in biological control agent populations using molecular data and crossbreeding experiments.

There are many examples of cryptic species that have been identified through DNA-barcoding or other genetic techniques. There are, however, very few confirmations of cryptic species being reproductively isolated. This study presents one of the few cases of cryptic species that has been confirmed to be reproductively isolated and therefore true species according to the biological species concept. The cryptic species are of special interest because they were discovered within biological control agent populations. Two geographically isolated populations of Eccritotarsus catarinensis (Carvalho) [Hemiptera: Miridae], a biological control agent for the invasive aquatic macrophyte, water hyacinth, Eichhornia crassipes (Mart.) Solms [Pontederiaceae], in South Africa, were sampled from the native range of the species in South America. Morphological characteristics indicated that both populations were the same species according to the current taxonomy, but subsequent DNA analysis and breeding experiments revealed that the two populations are reproductively isolated. Crossbreeding experiments resulted in very few hybrid offspring when individuals were forced to interbreed with individuals of the other population, and no hybrid offspring were recorded when a choice of mate from either population was offered. The data indicate that the two populations are cryptic species that are reproductively incompatible. Subtle but reliable diagnostic characteristics were then identified to distinguish between the two species which would have been considered intraspecific variation without the data from the genetics and interbreeding experiments. These findings suggest that all consignments of biological control agents from allopatric populations should be screened for cryptic species using genetic techniques and that the importation of multiple consignments of the same species for biological control should be conducted with caution.