Genetic diversity of the Metarhizium anisopliae complex in Colima, Mexico, using microsatellites.

Metarhizium anisopliae is a complex of cryptic species with wide geographical distribution and versatile lifestyles. In this study, 45 isolates of the Metarhizium genus harbored in the "Colección de Hongos Entomopatógenos" of the "Centro Nacional de Referencia de Control Biológico" from different substrates, insect-host, and localities from Colima, Mexico, were phylogenetically identified using the 5'end of translation elongation factor 1-α (5'TEF) and intergenic nuclear region MzFG543igs. Seven species were recognized, M. acridum (n = 26), M. pemphigi (n = 1), and within the PARB and MGT clades: M. anisopliae (N = 7; sensu stricto: n = 2; sensu lato: n = 5), M. brunneum (n = 2), M. guizhouense (n = 2), M. pingshaense (n = 2), and M. robertsii (n = 5). Twenty-nine SSR markers were developed for M. acridum; according to the analysis of 12 polymorphic SSR loci, M. acridum showed low genetic diversity, revealing five genotypes with a dominant one (n = 21). Based on the analysis of 13 specific SSR loci, 14 genotypes were identified within the PARB and MGT clades. This study contributes to generating valuable information about the community structure and genotypic diversity of Metharhizum species in the state of Colima, Mexico.

Molecular characterization of Chrysoperla carnea (Neuroptera: Chrysopidae) from commercial insectaries in Mexico.

The larvae of the Chrysoperla carnea-group (Neuroptera: Chrysopidae) are recognized among the most effective larval predators of various phytophagous arthropods. Therefore, green lacewings are commonly grown by commercial insectaries and released as biological control agents. Previous work has shown that commercial laboratories frequently supply indeterminate species of the large C. carnea cryptic species complex. In Mexico, at least 20 biological control companies have commercialized the species C. carnea, but none of the products reared by those companies have been analyzed scientifically. Thus, the goal of this work was to molecularly characterize nine C. carnea populations from Mexican insectaries using the most efficient molecular markers available: the mitochondrial genes COI, COII, ND2, and ND5. Phylogenetic analysis demonstrated a unique mitochondrial haplotype in seven commercial insectaries showing 100% similarity to the reference specimen C. plorabunda E100. In contrast, we observed two and four different mitochondrial haplotypes of the carnea-group in two commercial insectaries. More precisely, three specimens possessed the mitochondrial haplotype of the species C. zastrowi, suggesting possible natural occurrence of this haplotype in Mexico. Consequently, this study demonstrated the need for an extensive survey of the different laboratories and insectaries producing C. carnea in Mexico, including unambiguous species identification by song recordings to confirm the species identity of the observed mitochondrial haplotypes.

Non-destructive DNA extraction methods for entomophagous insects with emphasis on biological control.

One of the major challenges in molecular analysis of arthropods, especially for natural enemies of insect pests, is the intact preservation of the specimens to be integrated into entomological collections. However, most of the DNA extraction protocols involve maceration of the tissue, avoiding the preservation of the original specimen. Two general methods were adapted into non-destructive DNA extraction protocols, DNeasy® Blood & Tissue Kit (A) and the CaCl2 lysis buffer method (B), while the potential of the method with the alkaline lysis buffer (HotSHOT; C) was evaluated for the first time on insect specimens. These protocols were assessed for the recovery of DNA from Ceraeochrysa valida, Tamarixia radiata, and Hippodamia convergens. Photographical records showed that morphological features of the specimens were preserved after the DNA extraction process. COI fragments were successfully amplified with method A (100%), B (77%), and C (88%), respectively. We conclude that these non-destructive DNA extraction methods avoid the destruction of tissue and preserve the original insects and their morphological characteristics for future reference.

High genetic diversity of the entomopathogenic fungus Beauveria bassiana in Colima, Mexico.

The entomopathogenic fungus Beauveria bassiana is used widely as a biological control agent against a wide range of insect pests globally. In this study, 44 Beauveria isolates from the state of Colima, Mexico harbored in the "Colección de Hongos Entomopatógenos" of the "Centro Nacional de Referencia de Control Biológico" and from different substrates, insect-hosts, and localities were characterized with molecular markers. All isolates were identified using a Bayesian phylogenetic analysis of translation elongation factor 1-α (TEF) and nuclear intergenic Bloc region. Forty-three isolates were identified as B. bassiana and grouped into two sub-clades, i.e., AFNEO_1 (n = 22; previously defined as a clade with African and Neotropical origin) and Bb clade (n = 21; closely associated with ex-type strain ARSEF 1564), and one isolate was identified as B. pseudobassiana. The fixation index (FST = 0.493) established the genetic differentiation between AFNEO_1 and Bb clades. High genotype richness and genetic diversity of AFNEO_1 and Bb clades were revealed in sequence analysis of Bloc region and SSR genotyping. Moreover, the AFNEO_1 and Bb clades were confirmed as two independent clonally structured assemblages. Finally, the AMOVA detected no significant association between any combination of substrate, insect-host or geographical origin. High genetic variation of B. bassiana in Colima, Mexico could suggest a functional diversity among isolates that may include those effective against a specific insect pest.

Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi.

Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/ß-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications.

Viability, purity, and genetic stability of entomopathogenic fungi species using different preservation methods.

Preservation methods for entomopathogenic fungi (EPF) require effective protocols to ensure uniform processes and to avoid alterations during storage. The aim of this study was to preserve Beauveria bassiana, Metarhizium acridum, M. anisopliae, M. rileyi, Isaria javanica, Hirsutella thompsonii, H. citriformis and Lecanicillium lecanii in mineral oil (MO), sterile water (SW), silica gel (SG), lyophilisation (L), ultracold-freezing at -70 °C, and cryopreservation at -196 °C. The viability and purity of the fungi were then verified: phenotypic characteristics were evaluated qualitatively at 6, 12 and 24 m. Genetic stability was tested by amplified fragment length polymorphisms (AFLP) analysis at 24 m. Of the eight species of EPF, three remained viable in SW, five in MO and L, six at -70 °C, seven in SG, and eight at -196 °C. No significant changes were observed in AFLP patterns at 24 m of storage. The most effective preservation methods for EPF were SG, L, -70 and -196 °C. Beauveria bassiana, M. acridum, M. anisopliae, M. rileyi and I. javanica remained stable with all methods, while the remaining species were less compatible. The optimisation of preservation methods for EPF facilitates the development of reliable protocols to ensure their inherent characteristics in culture collections.

Species clarification of Isaria isolates used as biocontrol agents against Diaphorina citri (Hemiptera: Liviidae) in Mexico.

Entomopathogenic fungi belonging to the genus Isaria (Hypocreales: Cordycipitaceae) are promising candidates for microbial control of insect pests. Currently, the Mexican government is developing a biological control program based on extensive application of Isaria isolates against Diaphorina citri (Hemiptera: Liviidae), a vector of citrus huanglongbing disease. Previous research identified three promising Isaria isolates (CHE-CNRCB 303, 305, and 307; tentatively identified as Isaria fumosorosea) from Mexico. The goal of this work was to obtain a complete morphological and molecular characterization of these isolates. Comparative analysis of morphology established that the isolates showed similar characteristics to Isaria javanica. Multi-gene analysis confirmed the morphological identification by including the three isolates within the I. javanica clade. Additionally, this work demonstrated the misidentifications of three other Isaria isolates (CHE-CNRCB 310 and 324: I. javanica, formerly I. fumosorosea; CHE-CNRCB 393: I. fumosorosea, formerly Isaria farinosa), underlying the need for a full and correct characterization of an isolate before developing a biological control program. Finally, the inter-simple sequence repeat (ISSR) genotyping method revealed that the CHE-CNRCB 303, 305, and 307 isolates belong to three different genotypes. This result indicates that ISSR markers could be used as a tool to monitor their presence in field conditions.

Biological pest control in Mexico.

Mexico is a megadiverse country that forms part of the Mesoamerican biological corridor that connects North and South America. Mexico's biogeographical situation places it at risk from invasive exotic insect pests that enter from the United States, Central America, or the Caribbean. In this review we analyze the factors that contributed to some highly successful past programs involving classical biological control and/or the sterile insect technique (SIT). The present situation is then examined with reference to biological control, including SIT programs, targeted at seven major pests, with varying degrees of success. Finally, we analyze the current threats facing Mexico's agriculture industry from invasive pests that have recently entered the country or are about to do so. We conclude that despite a number of shortcomings, Mexico is better set to develop biological control-based pest control programs, particularly on an area-wide basis, than many other Latin American countries are. Classical and augmentative biological control and SIT-based programs are likely to provide effective and sustainable options for control of native and exotic pests, particularly when integrated into technology packages that meet farmers' needs across the great diversity of production systems in Mexico.

Environmental factors and interval from the introduction of rams to estrus in postpartum Saint Croix sheep.

The objective of this study was to determine whether season and total rainfall during the breeding season, sheep parity, and/or litter size affect the interval from the introduction of rams to estrus (IRE) in hair Saint Croix sheep in northeastern Mexico. An analysis was made of 874 services performed during 9 years, introducing the rams into the sheep flock after an isolation period of 60 days during the postpartum period. Estrus was recorded twice daily during 35 days. Year and season influenced significantly on the interval between ram introduction and estrus length (P < 0.01). Winter IRE length (7.9 ± 0.1 days) was shorter than in the other three seasons (11.1 ± 0.2, 11.1 ± 0.1, and 16.2 ± 0.2 days in summer, autumn, and spring, respectively) (P < 0.01). An interaction was observed between rainfall and season, then by rainfall between 0 and 100 mm, IRE was shorter (P < 0.05) in winter (6.8 ± 0.3 days), and by rainfall between 100 and 199 mm, IRE was shorter (P < 0.05) in autumn (10.2 ± 0.5 days); however, when rainfall was beyond 200 mm, IRE length was shorter (P < 0.01) in summer (4.8 ± 0.5 days) than in autumn (14.5 ± 0.3 days). The IRE length was also longer in first lambing ewes (P < 0.05) and was not affected by litter size. In the present study, several factors, including the breeding season, rainfall and parity, directly influenced the interval between the male introduction and the onset of estrus by Saint Croix hair sheep.