Prevalence and infection intensity of the biocontrol agent Paranosema locustae (Microsporidia) in field-collected,newly-associated hosts (Orthoptera: Acrididae: Melanoplinae)

Host range, prevalence, and infection intensity of Paranosema locustae in grasshoppers at an establishment site in Patagonia, Argentina, were recorded. Results agreed with earlier observations at other introduction-establishment areas. Affected grasshoppers were melanoplines (Baeacris punctulatus, Dichroplus elongatus, Dichroplus maculipennis). Sporulation was not observed in instars I, II, and III.     

Effect of Paranosema locustae (Microsporidia) on the behavioural phases of Locusta migratoria (Orthoptera: Acrididae) in the laboratory

The ability of parasites to modify the behaviour of their hosts is a wide spread phenomenon, but the effects of microsporidian parasites on locust behaviour remain unexplored. Here the frequencies of directional changes (ND) and jumping (NJ) per minute of gregarious locusts infected with 2000 spores of the microsporidian parasite Paranosema locustae were significantly different from those of untreated locusts 10 and 16 days after infection, being similar to values for solitary nymphs. In contrast, the behaviour of locusts inoculated with the lower doses of 200 spores/locust was sometimes like that of solitary nymphs. At other times, behaviour was intermediate between solitary and gregarious, i.e. transitional. The rearing density did not affect the turning and jumping behaviour of infected locusts, and their behaviours were similar to those of solitary locusts at 10–16 days after infection. Our study demonstrates that infection with P. locustae may lead gregarious locusts to change some of their behaviour to that typical of solitary locusts.     

Survival and fecundity of Dichroplus maculipennis and Ronderosia bergi (Orthoptera: Acrididae: Melanoplinae) following infection by Beauveria bassiana (Ascomycota: Hypocreales) under laboratory conditions

This study examined the effects of strain Beauveria bassiana (LPSC 1067) on nymphal development time, fecundity and adult survival in Dichroplus maculipennis and Ronderosia bergi under laboratory conditions. It was observed that infection with 1×10³ conidia/ml altered nymphal development time, fecundity and adult survival in both species. Mortality of D. maculipennis during third through the last instar (sixth) was significantly higher among treated nymphs (66±3.8%) than in controls (15±1.7%). Similarly, mortality in R. bergi during third through the last instar (fifth) was higher in treated nymphs (71±2.8%) than in controls (19±1.5%). Nymphal development times of both infected D. maculipennis and R. bergi were longer than controls. On the other hand, among survivors of both the species, control adults lived longer than infected adults. Finally, control grasshoppers of both species were much more successful reproductively than infected grasshoppers.     

Paranosema locustae (Microsporidia) in grasshoppers (Orthoptera: Acridoidea) of Argentina: field host range expanded

Detection of Paranosema locustae in three grasshopper species (Ronderosia bergi, Sinipta dalmani, Trimerotropis pallidipenis) within the establishment area of the Pampas expands the field host range of the pathogen to 20 species in the country. The newly recorded hosts might be important in the persistence and dispersal of the pathogen.     

Effect of Paranosema (Nosema) locustae (Microsporidia) on morphological phase transformation of Locusta migratoria manilensis (Orthoptera: Acrididae)

Field and laboratory studies demonstrated that Paranosema (Nosema) locustae had significant effects on the morphological phase transformation of Locusta migratoria manilensis (Meyen 1835). In the field, spraying P. locustae on gregarious locusts caused a substantial population reduction by 16 days after treatment, with most of the surviving locusts being phase solitaria. However, the effects of P. locustae on locust phase transformation began before direct mortality had caused a substantial reduction in locust density: locust numbers were still high at day 10, but locusts had already transformed to phase transiens. Laboratory assays showed that while a low dose of P. locustae had no effect on phase transformation, at a higher dose of 1×10⁵ spores/mL, locusts had F/C ratios that were significantly (P<0.05) more solitaria than untreated locusts, with locusts having ratios that were either phase solitaria or on the solitaria side of phase transiens. In a second laboratory experiment that analysed the effects of locust density on phase transformation by P. locustae, there was no obvious effect of density on female locusts 10 days later as all were solitaria at all locust densities. At day 16, female locusts were transiens at higher densities, but were solitaria at 4/cage. With males there were lesser effects. These results provide new evidence for P. locustae having sub-lethal effects on locust phase transformation at a wide range of locust densities.     

New case of long-term persistence of Paranosema locustae (Microsporidia) in melanopline grasshoppers (Orthoptera: Acrididae: Melanoplinae) of Argentina

We report an additional case of long-term persistence of Paranosema locustae in grasshoppers of Argentina. The pathogen was introduced from North America on rangeland at Loncopué, Neuquén province. Microsporidia were not detected in pre-introduction samples whereas infected grasshoppers were found 11 years after introduction. Affected grasshoppers were the melanoplines Dichroplus elongatus, Dichroplus maculipennis, and Scotussa lemniscata, some of them with high spore loads. The case highlights the ability of P. locustae to recycle in local grasshopper communities by parasitizing susceptible species other than the natural hosts.     

Efficiency of food utilization by Dichroplus maculipennis (Orthoptera: Acrididae: Melanoplinae) on four crop plants under controlled conditions

The aim of this study was to analyze the food utilization efficiencies and the relative growth and consumption rates of different developmental stages and sexes of D. maculipennis under controlled conditions on wheat, oat, corn, and soybean plants, important crops in the Pampas region of Argentina. As expected from a polyphagous species, D. maculipennis was observed to consume all four of the plant species offered. Nevertheless, the consumption of both nymphs and adults was differentiated. Oat and wheat were more consumed than corn and soybean. Females presented higher consumption rates (384.6 ± 30.64 mg/individual/day) than males (278.71 ± 24.26 mg/individual/day). Adult females had the highest growth rate, followed by nymphs of the same sex, and then adult males. The highest values of ECI and ECD were obtained in soybean; females had higher values of food efficiencies than males, and nymphs had greater values than adults. In relation to this, soybean was the highest quality food; the amount of nitrogen present in soybean was approximately twice that found in the other species. The nutritional needs of D. maculipennis might have been satisfied by feeding on low quantities of soybean, which is, among the food offered, the most “nutritionally balanced food”.     

Postembryonic development and food consumption of Dichroplus elongatus Giglio-Tos and Dichroplus maculipennis (Blanchard) (Orhtoptera: Acrididae: Melanoplinae) under laboratory conditions

Dichroplus maculipennis (Blanchard) and D. elongatus Giglio-Tos are two of the most important melanoplines in Argentina, both ecologically and economically. The postembryonic development and forage loss (consumption of Bromus brevis Ness + fallen material) caused by older nymphs (instars IV, V, VI) and adults of both species were studied under controlled conditions (30oC, 14L:10D, 40% RH). Five nymphal instars were recorded in D. elongatus, and six in D. maculipennis. Total nymphal development was similar in both species (D. elongatus: 32 ± 0.70 days; D. maculipennis: 34.5 ± 0.37 days). Daily consumption increased from nymphal instars to pre-reproductive adult stage. In both species, pre-reproductive females had higher consumption rates than other stages considered (D. elongatus: 30.6 ± 0.56 mg dry weight/day; D. maculipennis: 48.7 ± 0.74 mg dry weight/day). In the reproductive stage, consumption decreased significantly in both sexes. When feeding, D. maculipennis let some plant material to drop, increasing total loss. The percentage of fallen material was greater in reproductive adults, representing 3.9% and 2.9% of the total daily loss for males and females, respectively. Females and males of D. maculipennis were heavier than those of D. elongatus (P < 0.05), and daily consumption was significantly higher (P < 0.05). Regardless sex and reproductive status, adults of D. maculipennis consumed 29.1 ± 0.64 mg dry weight/day on average, while one of D. elongatus 20.0 ± 0.3 mg dry weight/ day.     

Use of entomopathogenic fungi combined with biorational insecticides to control Dichroplus maculipennis (Orthoptera: Acrididae: Melanoplinae) under semi-field conditions

Grasshoppers are among the invertebrate herbivores that cause most economic losses in grasslands throughout Argentina’s Pampas and parts of Patagonia. Chemical insecticides remain the sole option for grasshopper control in this area, despite being of significant environmental concern. Our aim was to evaluate the efficacy of combinations between three entomopathogenic fungi strains (Beauveria bassiana LPSc 1067 and LPSc1082), and Metarhizium anisopliae (LPSc 907), two biorational insecticides (luphenuron and methoxyfenozide), and a new synthetic chemical pesticide (rynaxypyr) in the control of the pest grasshopper Dichroplus maculipennis under field cage conditions. Fungal strains used were adjusted to 1 × 10⁸, 1 × 10⁶ and 1 × 10⁴ conidia/ml. Insecticides were tested at three concentrations: the average concentration recommended for application in the field (100%), 50% of that level and finally 25%. Combinations of the insecticides with B. bassiana (LPSc 1067, LPSc 1082) and M. anisopliae (LPSc 907) caused higher mortality to D. maculipennis nymphs than any of the individual agents used alone. The three insecticides tested did not negatively affect the isolates of the two species of entomopathogenic fungi employed.     

Status of the alien pathogen Paranosema locustae (Microsporidia) in grasshoppers (Orthoptera: Acridoidea) of the Argentine Pampas

After experimental introductions from North America in 1978–1982, the biocontrol agent Paranosema locustae became established in grasshopper communities of the western Pampas region of Argentina. The use and establishment of P. locustae in Argentina constitute both a case of neoclassical or new association biological control (use of an alien species against native pests) and a case of pathogen pollution (anthropogenic introduction–establishment of an infectious disease in populations of native species). Since P. locustae is a multihost pathogen among grasshoppers, its presence in the western Pampas represents an additional factor disrupting grasshopper communities according to the differential susceptibility of each species and possibly threatening some species. Microscopic examination of 504 grasshopper samples (the mean number of individuals per sample = 185) belonging to 43 species from 93 localities throughout the Pampas revealed an establishment area of approximate 90,000 km² from about 35° North to 38° South and from 61° East to 65° West. Field infections by P. locustae have now been detected in 21 grasshopper species in the western Pampas. Susceptible species with geographic distributions mostly restricted to the establishment area and with numerically small populations, like the melanopline Scotussa daguerrei, are predicted to be the ones facing higher risks of negative impacts.     

Distribution of genetic variability in populations of two chromosomal races of Dichroplus pratensis (Melanoplinae, Acrididae) and their hybrid zone

We examined, through allozyme electrophoresis, the genetic structure of populations of the acridid grasshopper Dichroplus pratensis from two chromosomal races (Northern and Southern) and their hybrid zone in Argentina. No fixed alleles for any particular race were found, although genetic differentiation among parental races was significant (0 = 0.044, 95% CI: 0.004-0.068). Hybrid populations are genetically more similar to the Southern race (0 = 0.008, 95% CI: -0.005-0.018) than to Northern ones (0 = 0.018, 95% CI: 0.002-0.030). Differential viability or fertility of hybrids, or asymmetry in mating preferences in favour of one particular cross would cause a higher proportion of matings between hybrid individuals and those from the Southern race. This would explain the high genetic similarity between those groups, in spite of their geographical vicinity with northern race populations.     

Selection of Antonospora locustae (Protozoa: Microsporidae) with higher virulence against Locusta migratoria manilensis (Orthoptera:Acrididae)

Antonospora locustae is a microsporidian parasite of grasshopper insects that is used as a biological control agent. We report on laboratory selection of isolates from different regions with increased virulence. Bioassays were conducted against third instar nymphs of Locusta migratoria manilensis. AL2008L01 was originally imported from the USA in 1986, AL2008M01 was isolated from Melanoplus differentialis in USA and AL2008F01 was isolated from infected Fruhstorferiola tonkinensi collected in Guangdong, China. The results showed that all three isolates can infect the locust and that pathogenicity increased gradually with increased dose. The LD₅₀ values of the original isolates at the highest dose (5×10⁶ spores/nymph) were 19, 23 and 22 days and LD₅₀ values were 3.2×10⁵, 3.4×10⁶ and 0.7×10⁶ spores/g, respectively. After selecting for three generations, the virulence of all isolates increased significantly. LT_50s were reduced to 17, 20 and 21 days at the highest dose (5×10⁶ spores/nymph) and LD_50s were reduced to 1.4×10⁵, 2.5×10⁵ and 1.7×10⁵ spores/g.     

Meiosis and the Neo- XY system of Dichroplus vittatus (Melanoplinae, Acrididae): a comparison between sexes

The origin of neo-XY sex systems in Acrididae is usually explained through an X-autosome centric fusion, and the behaviour of the neo-sex chromosomes has been solely studied in males. In this paper we analysed male and female Dichroplus vittatus. The karyotype comprises 2n = 20 chromosomes including 9 pairs of autosomes and a sex chromosome pair that includes a large metacentric neo-X and a small telocentric neo-Y. We compared the meiotic behaviour of the sex bivalent between both sexes. Mean cell autosomal chiasma frequency was low in both sexes and slightly but significantly higher in males than in females. Chiasma frequency of females increased significantly when the sex-bivalent was included. Chiasma distribution was basically distal in both sexes. Behaviour of the neo-XY pair is complex as a priori suggested by its structure, which was analysed in mitosis and meiosis of diploid and polyploid cells. During meiosis, orientation of the neo-XY is highly irregular; only 21% of the metaphase I spermatocytes show standard orientation. In the rest of cells, the alternate or simultaneous activity of an extra kinetochore in the distal end of the short arm (XL) of the neo-X, determined unusual MI orientations and a high frequency of non-disjunction and lagging of the sex-chromosomes. In females, the neo-XX bivalent had a more regular behaviour but showed 17% asynapsis in the XL arm which, in those cases orientated its distal ends towards opposite spindle poles suggesting, again, the activity of a second kinetochore. The dicentric nature and the unstable meiotic behaviour of the sex neo-chromosomes of D. vittatus suggest a recent origin of the sex determination mechanism, with presumable adaptive advantages which could compensate their potential negative heterosis. Our observations suggest that the origin of the neo-sex system was a tandem fusion of two original telocentric X-chromosomes followed by another tandem fusion with the small megameric bivalent and a further pericentric inversion of the neo-X. The remaining autosomal homolog resulted in the neo-Y chromosome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Postembryonic development, fecundity and food consumption of Dichroplus exilis (Orthoptera: Acrididae) under controlled conditions

Dichroplus exilis is a widely distributed species in Southern South America. Although there have been reports of D. exilis as an agricultural pest, some recent observations suggest that the damage attributed to D. elongatus may actually have been caused by D. exilis. This study was conducted to determine the postembryonic life cycle stages, fertility and food consumption of this species under controlled conditions (30 degrees C, 14L-10D, 40% RH). Individuals employed belong to the laboratory-hatched first generation (F1), from adults (n = 64, female = 28, male = 36) collected in natural grasslands near Rafaela, Santa Fe province in North-Eastern Argentina. Three cohorts of 16, 17 and 20 individuals were monitored independently in acetate tubes on a daily basis, until death of the last insect. Average fecundity was 381.84, 38.54 eggs per female. Egg-pod incubation time was 14.4, 1.08 days and six nymphal instars were recorded. Nymphal development time was 41.38, 0.71 days (I = 8.73, 0.20; II = 6.38, 0.24; III = 5.64, 0.33; IV = 7.15; 0.43; V=9.76, 0.54; IV = 7.85, 0.95). The recorded food consumption was 9.89, 1.08 (mg/ind/day) for nymphs IV, 18.04, 0.73 (mg/ind/day) for nymphs V-IV, 16.76, 1.06 (mg/ind/day) for pre-reproductive males, 28.09, 1.81 (mg/ind/day) for pre-reproductive females, 7.71,0.91 (mg/ind/day) for reproductive males and 13.06, 0.71 (mg/ind/day) for reproductive females, while the average adult food consumption, regardless of sex and reproductive status, was 16.41, 4.32 mg/day. Average food consumption of adult females was 17.47, 1.15 mg, and was significantly higher than that of males (10.83, 0.91mg). Data obtained in this study showed that D. exilis exhibits at least some of the biological attributes needed to configure an actual or potential agricultural pest, albeit not yet recognized as such. Field monitoring of grasshopper communities in areas where damage by D. exilis is suspected is envisaged in order to determine its possible status as a pest.     

Interaction between Paranosema locustae and Metarhizium anisopliae var. acridum, two pathogens of the desert locust, Schistocerca gregaria under laboratory conditions

The interaction between two pathogens, the microsporidian Paranosema locustae Canning and the fungus Metarhizium anisopliae var. acridum Driver and Milner was studied under laboratory conditions in an attempt to develop an improved method of microbial control for the desert locust, Schistocerca gregaria Forsk?l. Fifth-instar locust nymphs, reared in the laboratory, were treated with various concentrations of one of the two pathogens or with both pathogens. The numbers of locusts killed were recorded each day and the production of pathogen spores within the dead locusts was assessed at the end (day 21) of each experiment. Locust nymphs treated with both P. locustae and M. anisopliae died sooner than nymphs infected with only one of the pathogens. At the lower concentrations of pathogen tested, the effects of the two pathogens were additive. At the higher concentrations the combined effects were synergistic. In terms of locust mortality, there was no evidence of any antagonistic effects between the two pathogens. However, the production of spores by P. locustae was reduced considerably when the host insects were infected also with M. anisopliae. For example, nymphs treated initially with P. locustae and then treated 3 and 10 days later with M. anisopliae produced 3-20 times and 2.5-8 times fewer spores, respectively, than nymphs treated only with P. locustae. Hence, in areas where M. anisopliae is applied, the natural persistence of P. locustae in the local grasshopper and locust populations may be diminished.     

Influence of a rare unstable B-chromosome on chiasma frequency and nonhaploid sperm production in Dichroplus pratensis (Melanoplinae,Acrididae)

Three B-containing individuals of Dichroplus pratensis were found among 300 males collected in several Argentine localities. The B-chromosome is a partially euchromatic short telocentric and it is mitotically unstable. The B-chromosome number of testis cells ranged from 0 to 4 in two males and from 0 to 3 in the remaining one; this variation was inter- as well as intrafollicular. Meiotic behaviour of the Bs was very regular, association of Bs was usually chiasmate and no lagging univalents were observed during first division. The presence of Bs increased the production rate of abnormal spermatids and this effect was more pronounced in follicles with odd numbers of Bs. Chiasma frequency and between-cell variance were also increased in B-containing cells. A possible interaction between the Bs and the polymorphic centric fusions present in the species is discussed in relation to chiasma frequency determination.     

Interactions of two insect pathogens, Paranosema locustae (Protista: Microsporidia) and Metarhizium acridum (Fungi: Hypocreales), during a mixed infection of Locusta migratoria (Insecta: Orthoptera) nymphs

Locusta migratoria nymphs were fed Paranosema locustae spores and/or surface-treated with Metarhizium acridum 3 (assay 1), 6 (assay 2) or 9 days (assay 3) post microsporidia application (p.m.a.). These three dates corresponded to the key phases of P. locustae development: (a) mass proliferation, (b) transition to sporogenesis and (c) onset of spore maturation, respectively. As a result, locust mortality due to mixed treatment increased slower, equally and faster, as compared to mortality expected from the combination of two pathogens in assays 1-3, respectively. However, a statistically significant difference in survival times was observed only in assay 3, indicating that only at the phase of spore maturation microsporidia drastically increase locust susceptibility to fungal infection. Analysis of perished nymphs showed that fungal treatment 3 days p.m.a. impeded development of microsporidia. Fungal sporulation on locust cadavers was not affected by co-occurring microsporidiosis.     

Establishment of Liebermannia dichroplusae n. comb. on the basis of molecular characterization of Perezia dichroplusae Lange, 1987 (Microsporidia)

Perezia dichroplusae Lange, 1987 is a parasite of the Malpighian tubules of an Argentine grasshopper, Dichroplus elongatus (Orthoptera, Acrididae, Melanoplinae). In order to determine relationships of this microsporidium with Perezia nelsoni and with other microsporidia, we sequenced its small subunit ribosomal RNA gene (SSU rDNA) (GenBank Accession No. EF016249) and performed phylogenetic analysis of the novel sequence against 17 microsporidian SSU rDNA sequences from GenBank, using neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods. This analysis revealed the highest similarity (96%) of the new sequence to Liebermannia patagonica, a parasite of gut epithelium cells of another grasshopper from Argentina, versus only 65% similarity to P. nelsoni, a parasite of muscles of paenaeid shrimps. In phylogenetic trees inferred from SSU rDNA sequences, the microsporidium from D. elongatus is sister taxon to L. patagonica and both cluster with Orthosomella operophterae. At the higher hierarchical level, the Liebermania-Orthosomella branch forms a clade with the Endoreticulatus-Cystosporogenus-Vittaforma group and with Enterocytozoon bieneusi. Perezia nelsoni falls into another large clade together with Nosema and Ameson species. We propose transferring P. dichroplusae to the genus Liebermannia and creating a new combination Liebermannia dichroplusae n. comb., based both on SSU rDNA sequence analysis and on common characters between P. dichroplusae and L. patagonica, which include the presence of elongated multinuclear sporonts, sporoblastogenesis by a similar process of sequentially splitting off sporoblasts, ovocylindrical spores of variable size, tissue tropism limited to epithelial cells, Orthoptera as hosts, and geographical distribution of hosts in the southern temperate region of Argentina. We argue that the condition of the nuclei in spores (i.e. diplokaryotic in L. patagonica or monokaryotic in L. dichroplusae) cannot be used to distinguish genera. Therefore, we remove the statement about the presence of diplokaryotic spores from the revised diagnosis of the genus Liebermannia.     

Susceptibility of Bufonacris claraziana (Orthoptera: Tristiridae) to Beauveria bassiana (Ascomycota: Hypocreales) under controlled conditions

Bufonacris claraziana outbreaks, an endemic grasshopper of Argentinian Patagonia, are a recurrent phenomenon in that region, causing severe damage to forage. The aim of this study was to evaluate the susceptibility of B. claraziana to different strains of the entomopathogenic fungus Beauveria bassiana under laboratory conditions. The strains tested were LPSc 1225, LPSc 1226, and LPSc 1227. Percentage of mortality was 100% for all three strains, a significant difference was registered with the control where mortality was from 26.66?±?2.76%. Results obtained represent a first contribution on the use of an entomopathogenic microorganism as a potential biocontrol agent of this species.